The present invention is directed towards novel prodrugs of alcohol, amine, and thiol-containing compounds, to their preparation, to their synthetic intermediates, and to their uses. Prodrugs of the invention may be used to deliver drugs to the liver with high tissue specificity.
The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to be, or to describe, prior art to the invention. All cited publications are incorporated by reference in their entirety.
Drug induced toxicities and pharmacological side effects are often associated with interactions by the drug or drug metabolite in tissues not associated with the pharmacological benefits of the drug therapy. In other cases, the desired pharmacological effect is poorly achieved either because of dose-limiting toxicities or inadequate drug levels in the target tissues. Thus, there is a need to deliver drugs to specific tissues or organs. High organ specificity can be achieved by a variety of mechanisms including local administration to the target organ and drug-protein conjugates. Local administration to the target organ is an invasive procedure. Drug-protein conjugates exhibit poor oral bioavailability, limitations in carrier manufacturing and drug loading, a potential for diminished liver uptake due to down regulation of the receptor in diseased tissue, and a high incidence of antibody induction. A third approach entails use of prodrugs that are activated by enzymes highly enriched in the target organ.
There is particularly a need to deliver drugs to the liver to treat diseases such as hepatitis, cancer, malaria, and fibrosis which are poorly treated with current therapies. Many therapies for these conditions have narrow therapeutic indices. Other diseases such as hyperlipidemia where the liver is responsible for the overproduction of biochemical endproducts that directly contribute to the pathogenesis of disease can also be treated with liver specific drug delivery. Thus, there is still a need for prodrugs to enhance specificity.
The present invention is directed towards novel cyclic phosph(oramid)ate prodrugs of alcohol-, amine-, and thiol-containing drugs, their preparation, their synthetic intermediates, and their uses. Another aspect of the invention is the use of the prodrugs to treat diseases that benefit from enhanced drug distribution to the liver and like tissues and cells that express cytochrome P450, including hepatitis, cancer, liver fibrosis, malaria, other viral and parasitic infections, and metabolic diseases where the liver is responsible for the overproduction of the biochemical end product, e.g. glucose (diabetes); cholesterol, fatty acids and triglycerides (hyperlipidemia) (atherosclerosis) (obesity). In one aspect, the invention is directed towards the use of the prodrugs to enhance oral drug delivery. In another aspect, the prodrugs are used to prolong pharmacodynamic half-life of the drug. In addition, the prodrug methodology of the current invention is used to achieve sustained delivery of the parent drug. In another aspect, the prodrugs are used to increase the therapeutic index of the drug. In another aspect of the invention, a method of making these prodrugs is described. In another aspect, the prodrugs are also useful in the delivery of diagnostic imaging agents to the liver.
One aspect of the present invention concerns prodrugs of formula I 
wherein:
V, W, and Wxe2x80x2 are independently selected from the group consisting of xe2x80x94H, alkyl, aralkyl, alicyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, 1-alkenyl, and 1-alkynyl; or
together V and Z are connected via an additional 3-5 atoms to form a cyclic group containing 5-7 atoms, optionally 1 heteroatom, substituted with hydroxy, acyloxy, alkoxycarbonyloxy, or aryloxycarbonyloxy attached to a carbon atom that is three atoms from both Y groups attached to the phosphorus; or
together V and Z are connected via an additional 3-5 atoms to form a cyclic group, optionally containing 1 heteroatom, that is fused to an aryl group at the beta and gamma position to the Y attached to the phosphorus;
together V and W are connected via an additional 3 carbon atoms to form an optionally substituted cyclic group containing 6 additional carbon atoms and substituted with one substituent selected from the group consisting of hydroxy, acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy, and aryloxycarbonyloxy, attached to one of said additional carbon atoms that is three atoms from a Y attached to the phosphorus;
together Z and W are connected via an additional 3-5 atoms to form a cyclic group, optionally containing one heteroatom, and V must be aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
together W and Wxe2x80x2 are connected via an additional 2-5 atoms to form a cyclic group, optionally containing 0-2 heteroatoms, and V must be aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
Z is selected from the group consisting of xe2x80x94CHR2OH, xe2x80x94CHR2OC(O)R3, xe2x80x94CHR2OC(S)R3, xe2x80x94CHR2OC(S)OR3, xe2x80x94CHR2OC(O)SR3, xe2x80x94CHR2OCO2R3, xe2x80x94OR2, xe2x80x94SR2, xe2x80x94CHR2N3, xe2x80x94CH2aryl, xe2x80x94CH(aryl)OH, xe2x80x94CH(CHxe2x95x90CR22)OH, xe2x80x94CH(Cxe2x89xa1CR2)OH, xe2x80x94R2, xe2x80x94NR22, xe2x80x94OCOR3, xe2x80x94OCO2R3, xe2x80x94SCOR3, xe2x80x94SCO2R3, xe2x80x94NHCOR2, xe2x80x94NHCO2R3, xe2x80x94CH2NHaryl, xe2x80x94(CH2)pxe2x80x94OR12, and xe2x80x94(CH2)pxe2x80x94SR12;
p is an integer 2 or 3;
with the provisos that:
a) V, Z, W, Wxe2x80x2 are not all xe2x80x94H; and
b) when Z is xe2x80x94R2OR2, then at least one of V, W, and Wxe2x80x2 is not xe2x80x94H, alkyl, aralkyl, or alicyclic;
c) when Z is CHR2OH, then M is not xe2x80x94NH(lower alkyl), xe2x80x94N(lower alkyl)2, xe2x80x94NH(lower alkylhalide), xe2x80x94N(lower alkylhalide)2 or xe2x80x94N(lower alkyl)(lower alkylhalide); and
d) when V is aryl or substituted aryl, then M is not xe2x80x94O(D) where D is hydrogen, a metal ion or an ammonium ion;
R2 is selected from the group consisting of R3 and xe2x80x94H;
R3 is selected from the group consisting of alkyl, aryl, alicyclic, and aralkyl;
R6 is selected from the group consisting of xe2x80x94H, lower alkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl, and lower acyl;
R12 is selected from the group consisting of xe2x80x94H, and lower acyl;
each Y is independently selected from the group consisting of xe2x80x94Oxe2x80x94, and xe2x80x94NR6xe2x80x94;
M is selected from the group consisting of drugs MH containing an xe2x80x94OH, xe2x80x94NHR2, or xe2x80x94SH group, and that is attached to the phosphorus in formula I via O, N, or S of said OH, xe2x80x94NHR2, or SH group;
and pharmaceutically acceptable prodrugs and salts thereof.
Since these compounds have asymmetric centers, the present invention is directed not only to racemic and diastereomeric mixtures of these compounds, but also to individual stereoisomers. The present invention also includes pharmaceutically acceptable and/or useful salts of the compounds of formula I, including acid addition salts. The present inventions also encompass prodrugs of compounds of formula I.
In accordance with the present invention and as used herein, the following terms are defined with the following meanings, unless explicitly stated otherwise.
The term xe2x80x9carylxe2x80x9d refers to aromatic groups which have 5-14 ring atoms and at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. Suitable aryl groups include phenyl and furanyl.
Carbocyclic aryl groups are groups, wherein the ring atoms on the aromatic ring are carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and polycyclic or fused compounds such as optionally substituted naphthyl groups.
Heterocyclic aryl or heteroaryl groups are groups having from 1 to 4 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen. Suitable heteroaryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, and the like, all optionally substituted including bicyclic aromatics, e.g. benzimidazole.
The term xe2x80x9cbiarylxe2x80x9d refers to aryl groups containing more than one aromatic ring including both fused ring systems and aryl groups substituted with other aryl groups. Such groups may be optionally substituted. Suitable biaryl groups include naphthyl and biphenyl.
The term xe2x80x9calicyclicxe2x80x9d means compounds which combine the properties of aliphatic and cyclic compounds. Such cyclic compounds include but are not limited to, aromatic, cycloalkyl and bridged cycloalkyl compounds. The cyclic compound includes heterocycles. Cyclohexenylethyl and cyclohexylethyl are suitable alicyclic groups. Such groups may be optionally substituted.
The term xe2x80x9coptionally substitutedxe2x80x9d or xe2x80x9csubstitutedxe2x80x9d includes groups substituted by one to four substituents, independently selected from lower alkyl, lower aryl, lower aralkyl, lower alicyclic, hydroxy, lower alkoxy, lower aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, amino, guanidino, amidino, halo, lower alkylthio, oxo, acylalkyl, carboxy esters, carboxyl, -carboxamido, nitro, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, phosphono, sulfonyl, -carboxamidoalkylaryl, -carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy-, aminocarboxamidoalkyl-, cyano, lower alkoxyalkyl, lower perhaloalkyl, and arylalkyloxyalkyl. xe2x80x9cSubstituted arylxe2x80x9d and xe2x80x9csubstituted heteroarylxe2x80x9d preferably refers to aryl and heteroaryl groups substituted with 1-3 substituents. Preferably these substituents are selected from the group consisting of lower alkyl, lower alkoxy, lower perhaloalkyl, halo, hydroxy, and amino.
The term xe2x80x9caralkylxe2x80x9d refers to an alkylene group substituted with an aryl group. Suitable aralkyl groups include benzyl, picolyl, and the like, and may be optionally substituted. xe2x80x9cHeteroarylalkylxe2x80x9d refers to an alkylene group substituted with a heteroaryl group.
The term xe2x80x9calkylarylxe2x80x9d refers to an aryl group substituted with an alkyl group. xe2x80x9cLower alkylarylxe2x80x9d refers to such groups where alkyl is lower alkyl.
The term xe2x80x9clowerxe2x80x9d referred to herein in connection with organic radicals or compounds respectively defines such as with up to and including 10, preferably up to and including 6, and advantageously one to four carbon atoms. Such groups may be straight chain, branched, or cyclic.
The terms xe2x80x9carylaminoxe2x80x9d (a), and xe2x80x9caralkylaminoxe2x80x9d (b), respectively, refer to the group xe2x80x94NRRxe2x80x2 wherein respectively, (a) R is aryl and Rxe2x80x2 is hydrogen, alkyl, aralkyl or aryl, and (b) R is aralkyl and Rxe2x80x2 is hydrogen or aralkyl, aryl, alkyl.
The term xe2x80x9cacylxe2x80x9d refers to xe2x80x94C(O)R where R is alkyl or aryl.
The term xe2x80x9ccarboxy estersxe2x80x9d refers to xe2x80x94C(O)OR where R is alkyl, aryl, aralkyl, and alicyclic, all optionally substituted.
The term xe2x80x9ccarboxylxe2x80x9d refers to xe2x80x94C(O)OH.
The term xe2x80x9coxoxe2x80x9d refers to xe2x95x90O in an alkyl group.
The term xe2x80x9caminoxe2x80x9d refers to xe2x80x94NRRxe2x80x2 where R and Rxe2x80x2 are independently selected from hydrogen, alkyl, aryl, aralkyl and alicyclic, all except H are optionally substituted; and R and R1 can form a cyclic ring system.
The term xe2x80x9c-carboxylamidoxe2x80x9d refers to xe2x80x94CONR2 where each R is independently hydrogen or alkyl.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to xe2x80x94F, xe2x80x94Cl, xe2x80x94Br and xe2x80x94I.
The term xe2x80x9calkylaminoalkylcarboxyxe2x80x9d refers to the group alkyl-NR-alk-C(O)xe2x80x94Oxe2x80x94 where xe2x80x9calkxe2x80x9d is an alkylene group, and R is a H or lower alkyl.
The term xe2x80x9calkylxe2x80x9d refers to saturated aliphatic groups including straight-chain, branched chain and cyclic groups. Alkyl groups may be optionally substituted. Suitable alkyl groups include methyl, isopropyl, and cyclopropyl.
The term xe2x80x9ccyclic alkylxe2x80x9d or xe2x80x9ccycloalkylxe2x80x9d refers to alkyl groups that are cyclic of 3 to 10 atoms, more preferably 3 to 6 atoms. Suitable cyclic groups include norbornyl and cyclopropyl. Such groups may be substituted.
The term xe2x80x9cheterocyclicxe2x80x9d and xe2x80x9cheterocyclic alkylxe2x80x9d refer to cyclic groups of 3 to 10 atoms, more preferably 3 to 6 atoms, containing at least one heteroatom, preferably 1 to 3 heteroatoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen. Heterocyclic groups may be attached through a nitrogen or through a carbon atom in the ring. The heterocyclic alkyl groups include unsaturated cyclic, fused cyclic and spirocyclic groups. Suitable heterocyclic groups include pyrrolidinyl, morpholino, morpholinoethyl, and pyridyl.
The term xe2x80x9cphosphonoxe2x80x9d refers to xe2x80x94PO3R2, where R is selected from the group consisting of xe2x80x94H, alkyl, aryl, aralkyl, and alicyclic.
The term xe2x80x9csulphonylxe2x80x9d or xe2x80x9csulfonylxe2x80x9d refers to xe2x80x94SO3R, where R is H, alkyl, aryl, aralkyl, and alicyclic.
The term xe2x80x9calkenylxe2x80x9d refers to unsaturated groups which contain at least one carbon-carbon double bond and includes straight-chain, branched-chain and cyclic groups. Alkenyl groups may be optionally substituted. Suitable alkenyl groups include allyl. xe2x80x9c1-alkenylxe2x80x9d refers to alkenyl groups where the double bond is between the first and second carbon atom. If the 1-alkenyl group is attached to another group, e.g. it is a W substituent attached to the cyclic phosph(oramid)ate, it is attached at the first carbon.
The term xe2x80x9calkynylxe2x80x9d refers to unsaturated groups which contain at least one carbon-carbon triple bond and includes straight-chain, branched-chain and cyclic groups. Alkynyl groups may be optionally substituted. Suitable alkynyl groups include ethynyl. xe2x80x9c1-alkynylxe2x80x9d refers to alkynyl groups where the triple bond is between the first and second carbon atom. If the 1-alkynyl group is attached to another group, e.g. it is a W substituent attached to the cyclic phosph(oramid)ate, it is attached at the first carbon.
The term xe2x80x9calkylenexe2x80x9d refers to a divalent straight chain, branched chain or cyclic saturated aliphatic group.
The term xe2x80x9cacyloxyxe2x80x9d refers to the ester group xe2x80x94Oxe2x80x94C(O)R, where R is H, alkyl, alkenyl, alkynyl, aryl, aralkyl, or alicyclic.
The term xe2x80x9caminoalkyl-xe2x80x9d refers to the group NR2-alk- wherein xe2x80x9calkxe2x80x9d is an alkylene group and R is selected from H, alkyl, aryl, aralkyl, and alicyclic.
The term xe2x80x9calkylaminoalkyl-xe2x80x9d refers to the group alkyl-NR-alk- wherein each xe2x80x9calkxe2x80x9d is an independently selected alkylene, and R is H or lower alkyl. xe2x80x9cLower alkylaminoalkyl-xe2x80x9d refers to groups where each alkylene group is lower alkylene.
The term xe2x80x9carylaminoalkyl-xe2x80x9d refers to the group aryl-NR-alk- wherein xe2x80x9calkxe2x80x9d is an alkylene group and R is H, alkyl, aryl, aralkyl, and alicyclic. In xe2x80x9clower arylaminoalkyl-xe2x80x9d, the alkylene group is lower alkylene.
The term xe2x80x9calkylaminoaryl-xe2x80x9d refers to the group alkyl-NR-aryl- wherein xe2x80x9carylxe2x80x9d is a divalent group and R is H, alkyl, aralkyl, and alicyclic. In xe2x80x9clower alkylaminoaryl-xe2x80x9d, the alkylene group is lower alkyl.
The term xe2x80x9calkoxyaryl-xe2x80x9d refers to an aryl group substituted with an alkyloxy group. In xe2x80x9clower alkyloxyaryl-xe2x80x9d, the alkyl group is lower alkyl.
The term xe2x80x9caryloxyalkyl-xe2x80x9d refers to an alkyl group substituted with an aryloxy group.
The term xe2x80x9caralkyloxyalkyl-xe2x80x9d refers to the group aryl-alk-O-alk- wherein xe2x80x9calkxe2x80x9d is an alkylene group. xe2x80x9cLower aralkyloxyalkyl-xe2x80x9d refers to such groups where the alkylene groups are lower alkylene.
The term xe2x80x9calkoxyxe2x80x9d or xe2x80x9calkyloxy-xe2x80x9d refers to the group alkyl-Oxe2x80x94.
The term xe2x80x9calkoxyalkyl-xe2x80x9d or xe2x80x9calkyloxyalkyl-xe2x80x9d refer to the group alkyl-O-alk- wherein xe2x80x9calkxe2x80x9d is an alkylene group. In xe2x80x9clower alkoxyalkyl-xe2x80x9d, each alkyl and alkylene is lower alkylene.
The terms xe2x80x9calkylthioxe2x80x9d and xe2x80x9calkylthio-xe2x80x9d refer to the groups alkyl-Sxe2x80x94.
The term xe2x80x9calkylthioalkyl-xe2x80x9d refers to the group alkyl-S-alk- wherein xe2x80x9calkxe2x80x9d is an alkylene group. In xe2x80x9clower alkylthioalkyl-xe2x80x9d each alkyl and alkylene is lower alkylene.
The term xe2x80x9calkoxycarbonyloxy-xe2x80x9d refers to alkyl-Oxe2x80x94C(O)xe2x80x94Oxe2x80x94.
The term xe2x80x9caryloxycarbonyloxy-xe2x80x9d refers to aryl-Oxe2x80x94C(O)xe2x80x94Oxe2x80x94.
The term xe2x80x9calkylthiocarbonyloxy-xe2x80x9d refers to alkyl-Sxe2x80x94C(O)xe2x80x94Oxe2x80x94.
The terms xe2x80x9camidoxe2x80x9d or xe2x80x9ccarboxamidoxe2x80x9d refer to NR2xe2x80x94C(O)xe2x80x94 and RC(O)xe2x80x94NR1xe2x80x94, where R and R1 include H, alkyl, aryl, aralkyl, and alicyclic. The term does not include urea, xe2x80x94NRxe2x80x94C(O)xe2x80x94NRxe2x80x94.
The term xe2x80x9ccarboxamidoalkylarylxe2x80x9d and xe2x80x9ccarboxamidoarylxe2x80x9d refers to an aryl-alk-NR1xe2x80x94C(O), and ar-NR1xe2x80x94C(O)-alk-, respectively where xe2x80x9carxe2x80x9d is aryl, xe2x80x9calkxe2x80x9d is alkylene, R1 and R include H, alkyl, aryl, aralkyl, and alicyclic.
The term xe2x80x9chydroxyalkylxe2x80x9d refers to an alkyl group substituted with one xe2x80x94OH.
The term xe2x80x9chaloalkylxe2x80x9d refers to an alkyl group substituted with one halo.
The term xe2x80x9ccyanoxe2x80x9d refers to xe2x80x94Cxe2x89xa1N.
The term xe2x80x9cnitroxe2x80x9d refers to xe2x80x94NO2.
The term xe2x80x9cacylalkylxe2x80x9d refers to an alkyl-C(O)-alk-, where xe2x80x9calkxe2x80x9d is alkylene.
The term xe2x80x9caminocarboxamidoalkyl-xe2x80x9d refers to the group NR2xe2x80x94C(O)xe2x80x94N(R)-alk- wherein R is an alkyl group or H and xe2x80x9calkxe2x80x9d is an alkylene group. xe2x80x9cLower aminocarboxamidoalkyl-xe2x80x9d refers to such groups wherein xe2x80x9calkxe2x80x9d is lower alkylene.
The term xe2x80x9cheteroarylalkylxe2x80x9d refers to an alkyl group substituted with a heteroaryl group.
The term xe2x80x9cperhaloxe2x80x9d refers to groups wherein every Cxe2x80x94H bond has been replaced with a C-halo bond on an aliphatic or aryl group. Suitable perhaloalkyl groups include xe2x80x94CF3 and xe2x80x94CFCl2.
The term xe2x80x9cguanidinoxe2x80x9d refers to both xe2x80x94NRxe2x80x94C(NR)xe2x80x94NR2 as well as xe2x80x94Nxe2x95x90C(NR2)2 where each R group is independently selected from the group of xe2x80x94H, alkyl, alkenyl, alkynyl, aryl, and alicyclic, all except xe2x80x94H are optionally substituted.
The tern xe2x80x9camidinoxe2x80x9d refers to xe2x80x94C(NR)xe2x80x94NR2 where each R group is independently selected from the group of xe2x80x94H, alkyl, alkenyl, alkynyl, aryl, and alicyclic, all except xe2x80x94H are optionally substituted.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d includes salts of compounds of formula I and its prodrugs derived from the combination of a compound of this invention and an organic or inorganic acid or base. Suitable acids include HCl.
The term xe2x80x9cprodrugxe2x80x9d as used herein refers to any compound that when administered to a biological system generates a biologically active compound as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), and/or metabolic chemical reaction(s), or a combination of each. Standard prodrugs are formed using groups attached to functionality, e.g. HOxe2x80x94, HSxe2x80x94, HOOCxe2x80x94, R2Nxe2x80x94, associated with the drug, that cleave in vivo. Standard prodrugs include but are not limited to carboxylate esters where the group is alkyl, aryl, aralkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl as well as esters of hydroxyl, thiol and amines where the group attached is an acyl group, an alkoxycarbonyl, aminocarbonyl, phosphate or sulfate. The groups illustrated are exemplary, not exhaustive, and one skilled in the art could prepare other known varieties of prodrugs. Such prodrugs of the compounds of formula I, fall within the scope of the present invention. Prodrugs must undergo some form of a chemical transformation to produce the compound that is biologically active or is a precursor of the biologically active compound. In some cases, the prodrug is biologically active, usually less than the drug itself, and serves to improve drug efficacy or safety through improved oral bioavailability, pharmacodynamic half-life, etc. The biologically active compounds include, for example, anticancer agents, antiviral agents, and antibiotic agents.
The term xe2x80x9cbidentatexe2x80x9d refers to an alkyl group that is attached by its terminal ends to the same atom to form a cyclic group. For example, propyleneamine contains a bidentate propylene group.
The structure 
has a plane of symmetry running through the phosphorus-oxygen double bond when R6xe2x95x90R6, Vxe2x95x90W, Wxe2x80x2xe2x95x90H, and V and W are either both pointing up or both pointing down. The same is true of structures where each xe2x80x94NR6xe2x80x94 is replaced with xe2x80x94Oxe2x80x94.
The term xe2x80x9ccyclic 1xe2x80x2,3xe2x80x2-propane esterxe2x80x9d, xe2x80x9ccyclic 1,3-propane esterxe2x80x9d, xe2x80x9ccyclic 1xe2x80x2,3xe2x80x2-propanyl esterxe2x80x9d, and xe2x80x9ccyclic 1,3-propanyl esterxe2x80x9d refers to the following: 
The phrase xe2x80x9ctogether V and Z are connected via an additional 3-5 atoms to form a cyclic group containing 5-7 atoms, optionally containing 1 heteroatom, substituted with hydroxy, acyloxy, alkoxycarbonyloxy, or aryloxycarbonyloxy attached to a carbon atom that is three atoms from both Y groups attached to the phosphorusxe2x80x9d includes the following: 
The structure shown above (left) has an additional 3 carbon atoms that forms a five member cyclic group. Such cyclic groups must possess the listed substitution to be oxidized.
The phrase xe2x80x9ctogether V and Z are connected via an additional 3-5 atoms to form a cyclic group, optionally containing one heteroatom, said acyclic group is fused to an aryl group attached at the beta and gamma position to the Y adjacent to the phosphorusxe2x80x9d includes the following: 
The phrase xe2x80x9ctogether V and W are connected via an additional 3 carbon atoms to form an optionally substituted cyclic group containing 6 carbon atoms and substituted with one substituent selected from the group consisting of hydroxy, acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy, and aryloxycarbonyloxy, attached to one of said additional carbon atoms that is three atoms from a Y attached to the phosphorusxe2x80x9d includes the following: 
The structure above has an acyloxy substituent that is three carbon atoms from a Y, and an optional substituent, xe2x80x94CH3, on the new 6-membered ring. There has to be at least one hydrogen at each of the following positions: the carbon attached to Z; both carbons alpha to the carbon labeled xe2x80x9c3xe2x80x9d; and the carbon attached to xe2x80x9cOC(O)CH3xe2x80x9d above.
The phrase xe2x80x9ctogether W and Wxe2x80x2 are connected via an additional 2-5 atoms to form a cyclic group, optionally containing 0-2 heteroatoms, and V must be aryl, substituted aryl, heteroaryl, or substituted heteroarylxe2x80x9d includes the following: 
The structure above has V=aryl, and a spiro-fused cyclopropyl group for W and Wxe2x80x2.
The term xe2x80x9cphosph(oramid)itexe2x80x9d refers to phosphites, thiophosphites, and phosphoramidites which are compounds attached via O, S, or N, respectively, to the phosphorus in xe2x80x94P(YR)(YR) including cyclic forms, where Y is independently xe2x80x94Oxe2x80x94 or xe2x80x94NR6xe2x80x94.
The term xe2x80x9cphosph(oramid)atexe2x80x9d refers to phosphates, thiophosphates, and phosphoramidates which are compounds attached via O, S, or N, respectively, to the phosphorus in xe2x80x94P(O)(YR)(YR), including cyclic forms, where Y is independently xe2x80x94Oxe2x80x94 or xe2x80x94NR6xe2x80x94.
The term xe2x80x9ccyclic phosph(oramid)atexe2x80x9d refers to 
The carbon attached to V must have a Cxe2x80x94H bond. The carbon attached to Z must also have a Cxe2x80x94H bond.
The term xe2x80x9cliverxe2x80x9d refers to liver and to like tissues and cells that contain the CYP3A4 isozyme or any other P450 isozyme found to oxidize the phosph(oramid)ate esters of the invention. Based on Example F, we have found that prodrugs of formula VI and VIII are selectively oxidized by the cytochrome P450 isoenzyme CYP3A4. According to DeWaziers et al. (J. Pharm. Exp. Ther., 253, 387-394 (1990)), CYP3A4 is located in humans in the following tissues (determined by immunoblotting and enzyme measurements):
Thus, xe2x80x9cliverxe2x80x9d more preferably refers to the liver, duodenum, jejunum, ileum, colon, stomach, and esophagus. Most preferably, liver refers to the liver organ.
The term xe2x80x9cenhancingxe2x80x9d refers to increasing or improving a specific property.
The term xe2x80x9cliver specificityxe2x80x9d refers to the ratio:       [          drug      ⁢              xe2x80x83            ⁢      or      ⁢              xe2x80x83            ⁢      a      ⁢              xe2x80x83            ⁢      drug      ⁢              xe2x80x83            ⁢      metabolite      ⁢              xe2x80x83            ⁢      in      ⁢              xe2x80x83            ⁢      liver      ⁢              xe2x80x83            ⁢      tissue        ]        [          drug      ⁢              xe2x80x83            ⁢      or      ⁢              xe2x80x83            ⁢      a      ⁢              xe2x80x83            ⁢      drug      ⁢              xe2x80x83            ⁢      metabolite      ⁢              xe2x80x83            ⁢      in      ⁢              xe2x80x83            ⁢      blood      ⁢              xe2x80x83            ⁢      or      ⁢              xe2x80x83            ⁢      another      ⁢              xe2x80x83            ⁢      tissue        ]  
as measured in animals treated with the drug or a prodrug. The ratio can be determined by measuring tissue levels at a specific time or may represent an AUC based on values measured at three or more time points.
The term xe2x80x9cincreased or enhanced liver specificityxe2x80x9d refers to an increase in the liver specificity ratio in animals treated with the prodrug relative to animals treated with the parent drug.
The term xe2x80x9cenhanced oral bioavailabilityxe2x80x9d refers to an increase of at least 50% of the absorption of the dose of the parent drug or prodrug(not of this invention) from the gastrointestinal tract. More preferably it is at least 100%. Measurement of oral bioavailability usually refers to measurements of the prodrug, drug, or drug metabolite in blood, tissues, or urine following oral administration compared to measurements following systemic administration.
The term xe2x80x9cparent drugxe2x80x9d refers to any compound which delivers the same biologically active compound. The parent drug form is MH and standard prodrugs of MH, such as esters.
The term xe2x80x9cdrug metabolitexe2x80x9d refers to any compound produced in vivo or in vitro from the parent drug, which can include the biologically active drug.
The term xe2x80x9cpharmacodynamic half-lifexe2x80x9d refers to the time after administration of the drug or prodrug to observe a diminution of one half of the measured pharmacological response. Pharmacodynamic half-life is enhanced when the half-life is increased by preferably at least 50%.
The term xe2x80x9cpharmacokinetic half-lifexe2x80x9d refers to the time after administration of the drug or prodrug to observe a diminution of one half of the drug concentration in plasma or tissues.
The term xe2x80x9ctherapeutic indexxe2x80x9d refers to the ratio of the dose of a drug or prodrug that produces a therapeutically beneficial response relative to the dose that produces an undesired response such as death, an elevation of markers that are indicative of toxicity, and/or pharmacological side effects.
The term xe2x80x9csustained deliveryxe2x80x9d refers to an increase in the period in which there is a prolongation of therapeutically-effective drug levels due to the presence of the prodrug.
The term xe2x80x9cbypassing drug resistancexe2x80x9d refers to the loss or partial loss of therapeutic effectiveness of a drug (drug resistance) due to changes in the biochemical pathways and cellular activities important for producing and maintaining the biologically active form of the drug at the desired site in the body and to the ability of an agent to bypass this resistance through the use of alternative pathways and cellular activities.
The term xe2x80x9cbiologically active drug or agentxe2x80x9d refers to the chemical entity that produces a biological effect. Thus, active drugs or agents include compounds which as MH are biologically active.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d refers to an amount that has any beneficial effect in treating a disease or condition.
The invention is directed to the use of new cyclic 1,3-propanyl phosph(oramid)ate esters which are converted to phosphate, phosphoramidate, or thiophosphate containing compounds by P450 enzymes found in large amounts in the liver and other tissues containing these specific enzymes. The phosphates, phosphoramidates and thiophosphates are then hydrolized (by alkaline phosphatase, for example) to produce the free hydroxy, amine, or thiol, respectively. This methodology can be applied to various drugs and to diagnostic imaging agents which contain xe2x80x94OH, xe2x80x94NHR2, or xe2x80x94SH functionality. In effect, this methodology provides a prodrug (cyclic 1,3-propanyl phosph(oramid)ate esters) of a prodrug (phosphate, phosphoramidate, or thiophosphate) of a drug (contains xe2x80x94OH, xe2x80x94NHR2 or xe2x80x94SH).
In another aspect of the invention, this prodrug methodology can also be used to prolong the pharmacodynamic half-life because the cyclic phosph(oramid)ates of the invention can prevent the action of enzymes which degrade the parent drug.
In another aspect of the invention, this prodrug methodology can be used to achieve sustained delivery of the parent drug because oxidation of the prodrugs depends on the substituents V, Z, W, and Wxe2x80x2. Prodrugs found to oxidize slowly but provide therapeutic drug levels, therefore result in sustained drug delivery.
The novel cyclic 1,3-propanylester methodology of the present invention may also be used to increase the distribution of a particular drug or imaging agent to the liver which contains abundant amounts of the P450 isozymes capable of oxidizing the cylic 1,3-propanylester of the present invention so that the free phosph(oramid)ate is produced. The phosph(oramid)ate is then dephosphorylated by alkaline phosphatase, for example, in the target tissue to produce the active drug. Accordingly, this prodrug technology should prove useful in the treatment of liver diseases or diseases where the liver is responsible for the overproduction of the biochemical end product such as glucose, cholesterol, fatty acids and triglycerides. Such diseases include viral and parasitic infections, liver cancer, liver fibrosis, diabetes, hyperlipidemia, and obesity. Such anti-diabetic agents do not include FBPase inhibitors.
In addition; the liver specificity of the prodrugs should also prove useful in the delivery of diagnostic agents to the liver. The prodrugs of the present invention may be used to help identify diseases of the liver. For example, a person with a normal liver receiving a prodrug of an imaging agent would show the parent compound in the whole liver. A patient with a non-P450 expressing metastasis would show an area that does not contain the imaging agent.
These specific P450 enzymes are also found in other specific tissues and cells, and thus this methodology may also be used to increase the delivery of these agents to those tissues. In particular, certain cancers express P450. For instance, many cancers of the colon, soft tissue carcinoma, and metastases of hepatomas express P450 enzymes including CYP3A4. Such cancers even when outside the liver can be treated using the present invention.
In another aspect of the invention, the characteristic that most of the cyclic phosph(oramid)ates of the present invention are metabolized in the liver to produce the drug containing hydroxy, amine, or thiol, can enable the use of the prodrug methodology of the present invention to increase the therapeutic index of various drugs which tend to have side effects related to the amount of the drug or its metabolites which are distributed in extrahepatic tissues.
In another aspect of the invention, the cyclic phosph(oramid)ate prodrugs can increase the oral bioavailability of the drugs.
These aspects are described in greater detail below.
Prodrug Cleavage Mechanism
The prodrugs of the current invention are simple, low molecular weight modifications of the drug which enable liver-selective drug delivery on the basis of the their sensitivity to liver-abundant enzymes. The prodrug cleavage mechanism is supported through studies such as that shown in Examples A, E, F, and H. Prodrugs of the invention exhibit good stability in aqueous solutions across a broad pH range and therefore do not undergo a chemical cleavage process to produce the parent drug or free phosph(oramid)ate of the parent drug. (Example O). In addition the prodrugs exhibit good stability in plasma and in the presence of alkaline phosphatase. (Example P). In contrast to the parent drug, the prodrugs are rapidly cleaved in the presence of liver microsomes from rats (Example F) and humans (Example E). The drug is also produced in freshly isolated rat hepatocytes where it is detected as the parent drug (Example A). The by-product of the cleavage reaction was identified to further confirm the cleavage mechanism. (Example H).
Possible specific enzymes involved in the cleavage process were evaluated through the use of known cytochrome P450 inhibitors (Example F). The studies indicate that the isoenzyme cytochrome CYP3A4 is responsible for the oxidation based on ketoconozole inhibition of drug formation. Inhibitors of cytochrome P450 family 1 and/or family 2 do not inhibit prodrug cleavage.
Parent drug M, is also detected in the liver following administration of drugs of formulae VI-VIII, shown below: 
Prodrugs of formulae VI, VII, and VIII are particularly preferred.
Analysis of the by-products indicates that prodrugs of formula VI generate arylvinyl ketones (Example H), whereas prodrugs of formula VIII generate phenol. The mechanism of cleavage could proceed by the following mechanisms. 
Although the esters in the invention are not limited by the above mechanisms, in general, each ester contains a group or atom susceptible to microsomal oxidation (e.g. alcohol, benzylic methine proton), which in turn generates an intermediate that breaks down to the parent compound in aqueous solution via xcex2-elimination of the phosph(oramid)ate.
Furthermore, although these specific prodrugs are cleaved by CYP3A4, other pro drugs in the class may be substrates for other P450s. Small changes in structure are known to influence substrate activity and P450 preference.
In one aspect, the invention is directed to compounds that are prodrugs of formula I 
wherein
V is selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl. This class of prodrugs, shown above as class (2), readily undergo P450 oxidation at the benzylic methine proton (the proton on the carbon to which V is attached). In fact, there must be a hydrogen geminal to V to undergo this oxidation mechanism. Because Z, W, and Wxe2x80x2 are not at the oxidation site in this class of prodrugs, a broad range of substituents are possible. In one aspect, it is preferred if Z is an electron donating group which will reduce the mutagenicity or toxicity of the arylvinyl ketone that is the by-product of the oxidation of this class of prodrug. Thus, in this aspect Z is xe2x80x94OR2, xe2x80x94SR2, or xe2x80x94NR22.
In another aspect of prodrugs where V is aromatic, it is more preferred when Z is xe2x80x94R2, xe2x80x94OR2, xe2x80x94SR2, or xe2x80x94NR22, xe2x80x94OCOR3, xe2x80x94OCO2R3, xe2x80x94SCOR3, SCO2R3, xe2x80x94NHCOR2, xe2x80x94NHCO2R3, xe2x80x94(CH2)pxe2x80x94OR12, and xe2x80x94(CH2)pxe2x80x94SR12. Particularly preferred are such compounds where Z is not xe2x80x94OR2, xe2x80x94SR2, or xe2x80x94NR22. Especially preferred is when Z is H.
In this class of prodrug, it is more preferred when Wxe2x80x2 and W are independently selected from the group consisting of xe2x80x94H, alkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl. In one aspect, it is preferred when Wxe2x80x2 is H, and W and V are the same, and V and W are cis to one another. In another aspect, it is preferred when Wxe2x80x2 is H and W and V are the same, and V and W are trans to one another. More preferred, is when V, W, and Wxe2x80x2 are H, as this provides the similiest type of prodrug.
In another aspect, the invention is directed to compounds that are prodrugs of formula I 
wherein together V and Z are connected via an additional 3-5 atoms to form a cyclic group, optionally containing 1 heteroatom, said cyclic group is fused to an aryl group at the beta and gamma position to the Y adjacent to V. This class of prodrugs is susceptible to P450 oxidation and oxidizes by a mechanism analogous to those of class (2) shown above where V is aromatic. The same W and Wxe2x80x2 groups as described above for class (2) prodrugs are suitable.
In another aspect, the invention is directed to compounds that are prodrugs of formula I 
wherein Z is selected from the group consisting of of xe2x80x94HR2OH, xe2x80x94CHR2OC(O)R3, xe2x80x94CHR2OC(S)R3, xe2x80x94CHR2OC(S)OR3, xe2x80x94CHR2OC(O)SR3, xe2x80x94CHR2OCO2R3, xe2x80x94SR2, xe2x80x94CHR2N3, xe2x80x94CH2aryl, xe2x80x94CH(aryl)OH, xe2x80x94CH(CHxe2x95x90CR22)OH, xe2x80x94CH(Cxe2x89xa1CR2)OH, and xe2x80x94CH2NHaryl. This class of prodrugs; shown above as class (3), readily undergo P450 oxidation because they have a hydroxyl or hydroxyl equivalent (e.g., xe2x80x94CHR2OC(O)R3, xe2x80x94CHR2N3) with an adjacent (geminal) acidic proton. Z groups may also readily undergo P450 oxidation because they have a benzylic methine proton or equivalent (e.g., xe2x80x94CH2aryl, xe2x80x94CH(CHxe2x95x90CR22)OH). Where Z is xe2x80x94SR2, it is believed that this is oxidized to the sulfoxide or sulfone which will enhance the beta-elimination step. Where Z is xe2x80x94CH2NHaryl, the carbon next to nitrogen is oxidized to produce a hemiaminal, which hydrolizes to the aldehyde (xe2x80x94C(O)H), as shown above for class (3).
Because V, W, and Wxe2x80x2 are not at the oxidation site in this class of prodrugs, a broad range of V, W, and Wxe2x80x2 substituents is possible. One preferred aspect has V, Wxe2x80x2, and W substituted such that there is a line of symmetry though the phosphorus-oxygen double bond. More preferred is when V, Wxe2x80x2, and W are H.
In another aspect, the invention is directed to compounds that are prodrugs of formula I 
wherein
together V and Z are connected via an additional 3-5 atoms to form a cylic group containing 5-7 atoms, optionally 1 heteroatom, substituted with hydroxy, acyloxy, alkoxycarbonyloxy, or aryloxycarbonyloxy attached to a carbon that is three atoms from both Y groups attached to the phosphorus. This class of prodrugs undergoes P450 oxidation and oxidizes by a mechanism analogous to those of class (3) described above. The broad range of Wxe2x80x2 and W groups are suitable. Most preferred is H.
In another aspect, the invention is directed to compounds that are prodrugs of formula VIII 
wherein Zxe2x80x2 is xe2x80x94OH, xe2x80x94OC(O)R3, xe2x80x94OCO2R3, or OC(O)SR3. This class of prodrugs, shown above as class (1), readily undergoes P450 oxidation because they have a hydroxyl or hydroxyl equivalent group with an adjacent (geminal) acidic proton.
Because D3 and D4 are not at the oxidation site, a broad range of D3 and D4 substituents are possible, as long as one of D3 and D4 is H. This enables the ultimate elimination that produces phenol.
Alternatively, cyclic phosphoramidates can serve as a prodrug since intermediate phosphoramidates can generate the intermediate phosph(oramid)ate by a similar mechanism. The phosph(oramid)ate (MP(O)(NH2)Oxe2x88x92) is then converted to the drug MH either directly or via the phosph(oramid)ate (Mxe2x80x94PO32xe2x88x92). 
Enhanced Selective Delivery of Agents to the Liver and Like Tissues
Delivery of a drug to the liver with high selectivity is desirable in order to treat liver diseases or diseases associated with the abnormal liver properties (e.g. diabetes, hyperlipidemia) with minimal side effects.
Analysis of the tissue distribution of CYP3A4 indicates that it is largely expressed in the liver (DeWaziers et al., J. Pharm. Exp. Ther. 253: 387 (1990)). Moreover, analysis of tissue homogenates in the presence of prodrugs indicates that only the liver homogenate cleaves the prodrug. Kidney, brain, heart, stomach, spleen, muscle, lung, and testes showed no appreciable cleavage of the prodrug (Example D).
Evidence of the liver specificity can also be shown in vivo after both oral and i.v. administration of the prodrugs as described in Example E. Administration of the etoposide prodrug, Compound 1.1, is expected to result in enhanced liver specificity relative to free etoposide.
The prodrugs described in this invention can be tailored such that the elimination step is fast and therefore the product is produced near the site of oxidation, which for these prodrugs is in the liver or other P450-expressing tissue/cells.
In some cases liver specificity will be achieved most optimally using prodrugs of highly reactive drugs, which after production, act locally at a fast rate relative to diffusion out of the liver.
Increased Therapeutic Index
The prodrugs of this invention can significantly increase the therapeutic index (xe2x80x9cTIxe2x80x9d) of certain drugs. In many cases, the increased TI is a result of the high liver specificity.
The prodrugs described in this invention can be tailored such that the elimination step is fast and therefore confines production of the phosph(oramid)ate to the liver. Prodrugs of formula VI-VII do not readily recyclize, since the carbonyl product is a ketone except when Z=CH2OH in formula VII. Ketones do not hydrate to a great extent ( less than 2%), nor do they necessarily undergo the same metabolism associated with the aldehyde.
Severe toxicities are associated with nearly all anticancer agents. Most of these toxicities are associated with extrahepatic drug exposure. In an effort to decrease these toxicities during treatment of primary or secondary liver cancers, drugs are sometimes administered directly into the hepatic artery (e.g. floxuridine, mitomycin, etoposide). The high liver specificity of the prodrugs in the current invention suggest that systemic side effects will be minimized by the novel prodrug approach.
Moreover, primary and secondary liver cancers are particularly resistant to both chemotherapy and radiotherapy. Although the mechanism for the resistance is not completely understood, it may arise from increased liver gene products that lead to rapid metabolism and/or export of chemotherapeutic agents. In addition, the liver, which is generally associated with xenobiotic metabolism and generation of cytotoxic intermediates, is equipped by nature with multiple protective mechanism so that damage from these intermediates are minimized. For example, the intracellular concentration of glutathione is very high in the liver relative to other tissues presumably so that intermediates capable of alkylating proteins and DNA are detoxified through a rapid intracellular reaction. Consequently, the liver may be resistant to chemotherapeutic agents because of these mechanisms and therefore require higher than normal concentrations of the oncolytic agent to achieve success. Higher liver concentrations require higher doses of the drug which commonly result in extrahepatic toxicities.
To circumvent these limitations, drugs are sometimes administered directly into the hepatic artery (e.g. floxuridine, mitomycin, etoposide). Increased response rates are observed with this route of administration presumably because higher liver drug levels are achieved without an increase in the severity of the drug associated toxicities.
Prodrugs of the current invention achieve similar improvement in the response rate relative to extra-hepatic toxicities. This improvement in the therapeutic index provides an alternative strategy for the treatment of liver diseases, e.g. primary and secondary liver cancers.
The high liver specificity of prodrug cleavage implies that the by-product of prodrug cleavage is also primarily produced in the liver. Accordingly, toxicities associated with the by-product are minimized since the by-product frequently undergoes rapid detoxification reactions that either eliminate or minimize by-product toxicity. For example, reactions between the by-product and compounds and/or proteins present in the hepatocytes (e.g. glutathione and the xcex1,xcex2-unsaturated olefin generated by prodrugs of formulae VI and VII). Moreover, enzymes present in the liver may also further transform the by-product into a non-toxic compound (e.g. oxidation and/or sulfation of phenol, or reduction of the xcex1,xcex2-unsaturated ketone, etc.). In addition, intramolecular reactions that involve cyclization reactions between a reactive group and the xcex1,xcex2-unsaturated carbonyl-containing compound generated by prodrugs of formulae VI and VII can minimize by-product toxicity.
The cytotoxicity of the prodrugs are readily evaluated using cell lines that lack P450 activity (e.g. CYP3A4 activity). (Example C).
Non-Mutagenic Prodrugs
Prodrugs of the invention are generated by a postulated mechanism involving an initial oxidation followed by a xcex2-elimination reaction. In some cases, e.g. certain prodrugs of formula VI and formula VII, the by-product of the reaction is an xcex1,xcex2-unsaturated carbonyl compound, e.g. vinyl phenyl ketone for prodrugs where V=Ph, Z, W and Wxe2x80x2=H. Compounds that react with nucleophiles via a Michael addition can lead to certain toxicities, (e.g. acrolein produces bladder toxicities) and mutagenic activity. The degree to which these activities limit the use of compounds of Formula VI is dependent on the severity of the toxicity and the indicated disease.
Improved analogs of formula I are readily discoverable using known assays that can test for the mutagenicity of the prodrugs and its by-product.
Prodrugs that produce non-toxic and non-mutagenic by-products are especially preferred for the treatment of chronic diseases (e.g. diabetes). Frequently, it is difficult to predict the mutagenic properties of a compound. For example, a number of acrylates have been shown to produce positive mutagenic responses as indicated by increased chromosome aberrations and micronucleus frequencies in cultured L5179Y mouse lymphoma cells (Dearfield et al., Mutagenesis 4, 381-393 (1989)). Other acrylates, however, are negative in this test (J. Tox. Envir. Health, 34, 279-296 (1991)) as well as in the Ames test and the CHO assay which measures newly induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus (Mutagenesis 6, 77-85 (1991)). Phenyl vinyl ketone lacks teratogenic activity in rat embryos in culture suggesting that it may not be mutagenic nor highly toxic (Teratology 39, 31-37 (1989)).
Since mutagenicity and toxicity are not highly predictable properties, non-mutagenic prodrugs of formula I and their associated by-products can be readily identified by conducting well known in vitro and in vivo assays. For example, compounds can be tested in non-mammalian cell assays such as the Ames test, a fluctuation test in Kl. pneumoniae, a forward mutation assay with S. typhimurium, a chromosome loss assay in Saccharomyces cerevisiae, or a D3 recombinogenicity assay in Saccharomyces cerevisiae. Compounds can also be tested in mammalian cell assays such as the mouse lymphoma cells assay (TK+/xe2x88x92heterozygotes of L5178Y mouse lymphoma cells), assays in Chinese hamster ovary cells (e.g. CHO/HGPRT assay), and an assay in rat liver cell lines (e.g. RL1 or RL4). Each of these assays can be conducted in the presence of activators (e.g. liver microsomes) which may be of particular importance to these prodrugs. By conducting these assays in the presence of the liver microsomes, for example, the prodrug produces products, such as phenol or vinyl ketone. The mutagenicity of the by-product is measured either directly or as a prodrug where the results are compared to the parent drug alone. In addition, the assays can be carried out in the absence of activators and with cell lines that lack P450 activity and thereby enable measurement of the cytotoxicity and mutagenicity of the prodrug. Assays in liver cell lines are a preferred aspect of the invention since these cells have higher glutathione levels, which can protect the cell from damage caused by a Michael acceptor, as well as greater levels of intracellular enzymes used to detoxify compounds. For example, the liver contains reductases that with some by-products might result in reduction of the carbonyl.
A variety of end points are monitored including cell growth, colony size, gene mutations, micronuclei formation, mitotic chromosome loss, unscheduled DNA synthesis, DNA elongation, DNA breaks, morphological transformations, and relative mitotic activity.
In vivo assays are also known that assess the mutagenicity and carcinogenicity of compounds. For example, a non-mammalian in vivo assay is the Drosophila sex-linked recessive lethal assay. Examples of mammalian in vivo assays include the rat bone marrow cytogenetic assay, a rat embryo assay, as well as animal teratology and carcinogenicity assays.
In some cases the prodrug substituents are selected such that the by-product produced is poor Michael acceptor. For example, for class (2) prodrugs (V is aromatic or the like) when Z in formula I is an electron donator, the corresponding arylvinylketone produced are less likely to act as a Michael acceptor. Similarly, when W, Wxe2x80x2 or both W and Wxe2x80x2 are not hydrogen (e.g. methyl or phenyl) Michael addition to the beta-carbon is greatly diminished.
Enhancing Oral Bioavailability
The invention pertains to certain cyclic 1xe2x80x2,3xe2x80x2-propanyl esters of phosph(oramid)ates and the use of these esters to deliver, most preferably via oral administration, a therapeutically effective amount of the corresponding drug containing a free xe2x80x94OH, xe2x80x94NHR2, or xe2x80x94SH, preferably to an animal in need thereof. Prodrugs of the invention enhance oral bioavailability of certain drugs by changing the physical properties of the drug as a consequence of the prodrug moiety and its substituents V, Z, W, and Wxe2x80x2. The active drug is referred to as MH.
Drugs containing amines are often protonated at physiological pH and are therefore poorly absorbed from the gastrointestinal tract. Prodrugs of the present invention attached to amines eliminate the charge and increase the overall hydrophobicity thereby enhancing absorption via passive diffusion processes. For example, many hydrophobic drugs are poorly absorbed because of very low water solubility. Prodrugs of these drugs, especially prodrugs that contain a weakly basic nitrogen (e.g. V=4-pyridine) greatly enhance water solubility. Water solubility is a particular problem with oncolytic drugs (e.g. taxol and taxol derivatives). Last, oral absorption can be limited by drug metabolism within the gastrointestinal tract, blood, or other extrahepatic organs, which can be prevented by a suitably positioned prodrug moiety.
The prodrugs of the invention exhibit improved properties that lead to enhanced oral bioavailability relative to the parent drug. Several characteristics of the present cyclic phosph(oramid)ate prodrugs may contribute to their ability to enhance oral bioavailability. First, the prodrugs exhibit good stability in aqueous solutions across a wide range of pHs. This pH stability prevents immediate hydrolysis in the mouth and GI tract prior to absorption. The pH stability can also be beneficial during formulation of the product.
Second, the prodrugs are resistant to esterases, phosphatases, and other non-oxidative enzymes (e.g. deaminases) that are abundant in the gastrointestinal tract. Because much of the administered dose remains intact in the G.I. tract, more of the drug can be absorbed by passive diffusion and enter the blood stream.
Last, the prodrug can limit metabolism at other sites on the molecule. For example, the prodrugs of the invention may eliminate metabolism by non-hepatic enzymes and thereby enable more of the drug to circulate in the blood stream for longer duration. Although not all of these properties will be applicable to every prodrug of every drug, each of these properties can enable more drug to survive the GI tract and be available for absorption.
Oral bioavailability can be calculated by comparing the area under the curve of prodrug, drug, and/or metabolite concentration over time in plasma, liver, or other tissue or fluid of interest following oral and i.v. administration. Oral bioavailability can often be measured by comparing the amount of the parent compound excreted in the urine, for example, after oral and i.v. administration of the prodrug. A lower limit of oral bioavailability can be estimated by comparison with the amount of parent drug excreted in the urine after administration of the prodrug (p.o.) and the prodrug or parent drug (i.v.). Prodrugs of the invention show improved oral bioavailability across a wide spectrum of prodrugs, with many preferably showing at least a 0.5-25-fold increase in oral bioavailability.
More preferably, oral bioavailability is enhanced by at least 2-fold compared to the parent drug.
Sustained Delivery
Drugs that undergo rapid elimination in vivo often require multiple administrations of the drug to achieve therapeutically-effective blood levels over a significant period of time. Alternative methods are available for this purpose including sustained release formulations and devices. Co-administration of compounds that block either the metabolism or elimination of the drug is another strategy. Prodrugs that breakdown over time can also provide a method for achieving sustained drug levels. In general, this property has not been possible with the known phosph(on)ate prodrugs since either they undergo rapid hydrolysis in vivo (e.g. acyloxyalkyl esters) or very slow conversion (e.g. di-aryl prodrugs).
The cyclic phosph(oramid)ates of the invention are capable of providing sustained drug release by providing a steady release of the drug over time. Suitably positioned prodrug moieties on the parent drug MH can prevent or slow systemic metabolism associated with the parent drug.
Sustained delivery of the drugs is achievable by selecting the prodrugs of formula I that are hydrolyzed in vivo at a rate capable of achieving therapeutically effective drug levels over a period of time. The cleavage rate of the drug may depend on a variety of factors, including the rate of the P450 oxidation, which is dependent on both the substituents on the prodrug moiety, the stereochemistry of these substituents and the parent drug. Moreover, sustained drug production will depend on the rate of elimination of the intermediate generated after oxidation and the availability of the prodrug to the liver, which is the major site of oxidation. Identification of the prodrug with the desired properties is readily achieved by screening the prodrugs in an assay that monitors the rate of drug production in the presence of the major P450 enzyme involved in the metabolism, in the presence of liver microsomes or in the presence of hepatocytes. These assays are illustrated in Examples B, and F, and A, respectively.
It is contemplated that prodrugs of the present invention could be combined to include, for example, one prodrug which produces the active agent rapidly to achieve a therapeutic level quickly, and another prodrug which would release the active agent more slowly over time.
Improved Pharmacodynamic Half-Life
The pharmacodynamic half-life of a drug can be extended by the novel prodrug methodology as a result of both its ability to produce drug over a sustained period and in some cases the longer pharmacokinetic half-life of the prodrug. Both properties can individually enable therapeutic drug levels to be maintained over an extended period resulting in an improvement in the pharmacodynamic half-life. The pharmacodynamic half-life can be extended by impeding the metabolism or elimination pathways followed by the parent drug. For some drugs, the prodrugs of the present invention are able to avoid the rapid metabolism or elimination pathways associated with the parent drug and thereby exist as the prodrug for extended periods in an animal. High levels of the prodrug for an extended period result in sustained production of the parent drug which can result in an improvement in the drug pharmacodynamic half-life.
Types of Parent Drugs
Various kinds of parent drugs can benefit from the prodrug methodology of the present invention. It is preferred that the prodrug phosph(oramid)ate moiety be attached to a hydroxy, amine, or thiol on the parent drug. In many cases the parent drug will have many such functional groups. The preferred group selected for attachment of the prodrug is the group that is most important for biological activity and is chemically suitable for attachment of the prodrug moiety. Thus, the phosph(oramid)ate moiety will prevent the prodrug from having biological activity. An inactive prodrug should limit systemic side effects because higher drug concentrations will be in the target organ (liver) relative to non-hepatic tissues. The amine should have at least one Nxe2x80x94H bond, and preferably two.
Treatment of Cancer
The prodrug strategy in the current invention encompasses several features that are advantageously used in cancer therapies. The prodrug strategy can be effective in the treatment of liver cancer because the drug is cleaved by liver-abundant enzymes which suggests that a greater therapeutic index will result since much less parent drug is present in the blood and therefore available to produce side effects arising from effects on cells in the blood or via distribution to other tissues.
Cancer cells that express P450s, especially CYP3A4, are sensitive to prodrugs of the invention since the prodrugs are cleaved inside these cells to produce high local concentrations of the drug relative to drug concentrations in other tissues/cells that lack CYP3A4 activity. Studies reported in the literature suggest that certain carcinomas and sarcomas express CYP3A4. For example, hepatocellular carcinomas exhibit approximately 50-100% of normal CYP3A4 activity. Cancers outside the liver may also exhibit CYP3A4 activity whereas normal tissue surrounding the tumor is devoid of activity. Tumors that metastasize to the liver from non-P450-expressing organs (e.g. breast) often do not have P450 activity. Prodrugs of the invention, however, are still suitable for treatment of these tumors since the drug is produced in normal hepatocytes and depending on the drug, can diffuse out of the hepatocyte and into the tumor. The highest concentrations of the drug are in the liver and therefore the exposure of the liver tumor to the drug is high relative to extra-hepatic organs. Examples of preferred drug candidates that are specifically amenable to the strategy include, e.g. etoposide, teniposide, and other epipodophyllotoxins (such as NK-611, Azatoxin, and GL-331); camptothecin, topotecan, irinotecan, lurtotecan, 9-aninocamptothecin, and other camptothecins (such as DX-8951F, GG-211, SKF 107874, SKF 108025); neocarzinostatin, calicheamicin, esperamicin, and other enediyne antibiotics; paclitaxel, docetaxel, and other taxanes (such as FCE-28161); coformycin, 2xe2x80x2-deoxycoformycin; doxorubicin; daunorubicin; idarubicin, pirarubicin, epirubicin, and other anthracycline glycosides; mytomycin; eflornithine, and other polyamine biosynthesis inhibitors; combrestatin, combretastatin, and other analogs; vinblastine, vincristine, vindesine, vinorelbine, and other Vinca alkaloids; mycophenolic acid and other IMPDH inhibitors; mitoxantrone, piroxantrone, losoxantrone, and other anthrapyrazoles.
Treatment of Viral Infections
Drugs useful for treating viruses that infect the liver and cause liver damage, e.g. hepatitis virus strains, exhibit similar properties to the anticancer drugs in terms of efficacy, side effects and resistance. Such drugs are not nucleosides that are active in the phosphorylated form. In some cases, the drugs are already targeted for hepatitis. The prodrugs of these compounds could enhance the efficacy, increase the therapeutic index, improve the pharmacodynamic half-life and/or bypass drug resistance. Prodrugs of other agents used to treat viral infections other than hepatitis may also be made useful by administration of the prodrugs of this invention since the resistance will be avoided by delivery to the liver.
Treatment of Liver Fibrosis
It is contemplated that the prodrug methodology of the present invention could be used for the delivery of drugs useful for the treatment of liver fibrosis. Liver fibrosis is characterized by the excessive accumulation of extracellular matrix proteins, primarily collagen, in the liver. Liver fibrosis results from liver injury and is associated with the accumulation and activation of a variety of inflammatory cells, including neutrophils, monocytes/macrophages and platelets. Release of cytokines and growth factors from these cells leads to the recruitment and proliferation of fibroblasts and other related cells that excrete ECM proteins. In the case of the liver, the hepatic stellate cell (Ito cell), is the principal effector. Once activated, these cells are transformed from cells that in the normal liver are responsible for storing fats to cells that have many features of smooth muscle cells and actively secrete proteins. The proteins produced by the stellate cell include five types of collagen, heparin sulfate, dermatan, chondroitin sulfate proteoglycans, laminin, cellular fibronectin, tenascin, decorin and biglycan. Production of the ECMs leads to a high-density matrix in the subendothelial space which leads to loss of hepatocytic microvilli and sinusoidal fenestrations. Activated stellate cells are known to express CYP3A4 and therefore may be sensitive to drugs delivered as prodrugs of this invention.
Many potential drugs for treating liver fibrosis are limited by side effects due to inhibition of fibrotic processes at extrahepatic sites (wounds, tendons, etc). Potential drugs that could benefit from the prodrug approach include endothelin receptor antagonists, prostaglandin E1/E2, retinoids analogs, corticosteroids, D-penicillanine, prolyl hydroxylase inhibitors, lysyl oxidase inhibitors, NF-kappaB transcription factor antagonists, cytokine production inhibitors, metalloproteinase inhibitors, and cytotoxic agents (mentioned above for cancer treatments).
Agents Used to Modulate CYP Activity
A variety of methods may be used to enhance the in vivo activity of compounds of formula I. For example, various drugs are known that enhance cytochrome P450 (CYP) activity. Enhancement frequently entails increased gene transcription. Four families of CYPs are particularly susceptible to induction, namely CYP1-4. Induction is purportedly via receptors that are activated by various xenobiotics. For example, CYP1 gene activation frequently involves activation of the Ah receptor by polycyclic aromatic hydrocarbons. CYP2-4 are activated via orphan nuclear receptors. Data suggests that the nuclear receptor CAR (constitutively Active Receptor) is responsible for phenobarbital CYP activation, especially CYP2 genes. The pregnane nuclear receptors (PXR or PAR or SXR) are thought to activate CYP3A genes whereas the PPAR (peroxisome proliferator activate receptor) is linked to CYP4 gene activation. All three xenobiotic receptors are highly expressed in the liver which accounts for the liver specificity of the P450 gene induction.
Xenobiotics known to induce CYP3 genes include phenobarbital, a variety of steroids, e.g. dexamethasone, antibiotics, e.g. rifampicin, and compounds such as pregnenolone-16a carbonitrile, phenytoin, carbamazepine, phenylbutazone, etc. A variety of methods are known that enable identification of xenobiotics that induce P450s, including a reporter gene assay in HepG2 cells (Ogg et al., Xenobiotica 29, 269-279 (1999). Other inducers of the CYP3A subfamily are known that act at the post-transcriptional level either by mRNA or protein stabilization, e.g. clotrimazole, TA and erythromycin. Compounds known to induce CYPs or identified in in vitro assays are then used to enhance CYP activity in vivo. For example, CYP activity is monitored in rats pre-treated with CYP modulators by e.g. evaluating liver microsomes over a period of time to determine the optimal pre-treatment period, dose and dosing frequency. Rats with enhanced CYP activity, especially the CYP activity responsible for activation of the prodrugs (e.g. CYP3A4), are then treated with compounds of formula 1. Enhanced CYP activity can then lead to enhanced prodrug conversion and liver specificity. For example, enhanced metabolism of cyclophosphamide was found with pre-treatment with phenobarbital (Yu et al., J. Pharm. Exp. Ther. 288, 928-937 (1999).
In some cases, enhanced CYP activity may lead to unwanted drug metabolism. For example, enhanced activity of CYPs not involved in prodrug activation can result in increased drug metabolism and therefore decreased efficacy. In addition, increased CYP activity in other tissues, e.g. CYP3A4 in the gastrointestinal tract, could result in decreased prodrug absorption and liver drug levels. Inhibitors of CYP activity are known that might be useful in minimizing unwanted drug metabolism. For example, grapefruit juice is known to inactivate gastrointestinal CYP3A4 and to result in enhanced absorption of numerous drugs metabolized by CYP3A4. CYP inhibitors are also known for many of the CYP subfamilies that can be useful for attenuating unwanted drug metabolism while maintaining CYP activity important for prodrug cleavage. For example, the CYP3A inhibitor TAO was used to modulate cyclophosphamide metabolism in vivo in a manner that decreased the formation of toxic metabolites that do not contribute to its antitumor activity.
Use for Treating Hepatocellular Carcinomas (HCC)
Oncolytic drugs such as etoposide, topotecan, taxol, etc. that contain a biologically important hydroxyl or oncolytic drugs such as mitomycin, anthracyclin antibiotics (e.g. doxorubicin) that contain a biologically important amino group or oncolytic drugs that contain a sulfhydryl moiety are suitable drugs for conversion to compounds of formula 1. These compounds are especially useful for the treatment of HCC since transformed cells contain abundant CYP3A4 activity. Furthermore it is known that delivery of 5-FU, taxol and other oncolytic agents to the liver via portal vein or intra-arterial infusion has shown significant success with response rates as high as 50% (normal rate is less than 20%). However, the complexity, expense and high incidence of secondary complications associated with long-term percutaneous catheterization or infusion devices diminishes the likelihood that local drug administration will become the standard therapy for liver cancer.(Venook, A. P. J. Clin. Oncol. 12, 1323-1334 (1994); b) Atiq, O. T.; Kemeny, N.; Niedzwiecki, D.; et al. Cancer, 69, 920-924 (1992).)
Use for Treating Secondary Liver Tumors
Oncolytic drugs such as etoposide, topotecan, taxol, etc. that contain a biologically important hydroxyl or oncolytic drugs such as mitomycin, methotrexate, anthracyclin antibiotics (e.g. doxorubicin) that contain a biologically important amino group or oncolytic drugs that contain a sulfhydryl moiety are suitable drugs for conversion to compounds of formula 1. Although these tumors often exhibit substantially lower CYP activity than normal tissues, antitumor effects are gained via selection of oncolytic agents that once produced in liver hepatocytes, diffuse out of the hepatocyte and enter the bloodstream as well as nearby cells. Since drug generation is in the liver, a greater concentration of the drug is found in nearby tissue than in extrahepatic tissue thereby leading to enhanced efficacy and an increased therapeutic index.
Use for Treating Extra-Hepatic Carcinomas
Oncolytic drugs such as etoposide, topotecan, taxol, etc. that contain a biologically important hydroxyl or oncolytic drugs such as mitomycin, methotrexate, anthracyclin antibiotics (e.g. doxorubicin) that contain a biologically important amino group or oncolytic drugs that contain a sulfhydryl moiety are suitable drugs for conversion to compounds of formula 1. In general, the CYP3 family of genes is expressed in normal tissues predominantly in the liver and gastrointestinal tract. CYP activity, however, is also known to be expressed in various soft tissue sarcomas and other cancers, possibly as part of a drug resistance mechanism. To date, CYP3 activity has been found in renal cancer, lung cancer, stomach cancer, breast cancer. Little tumor heterogeneity is observed (Murray et al., J. Pathology, 171, 49-52 (1993); Murray et al., British J. Cancer, 79, 1836-1842 (1999)). Accordingly, the prodrugs of formula 1 are useful for treating cancers in which CYP activity, particularly CYP3A, is present.
Methods for Monitoring Patient P450 Activity
CYP activity is known to exhibit significant differences across individuals. The range for CYP3A4 is 5- to 20-fold although most individuals are within a 3-fold range. Modest decreases are noted for individuals with liver disease (30-50%) or advanced age (25-50%). Differences for gender are even more modest( less than 25%). Methods for phenotyping an individual""s CYP activity are known and could be useful in predicting who should receive drugs that modulate CYP activity. Evasive procedures include liver biopsy. Non evasive procedures have been reported, including an xe2x80x9cerythromycin breath testxe2x80x9d which is based on the exhalation of 14CO2 generated from the CYP3A-mediated N-demethylation of radiolabeled erythromycin (iv). (Watkins, Pharmacogenetics 4, 171-184 (1994)).
Gene Therapy
Introduction into tumor cells genes that encode for enzymes not normally expressed represents a new therapeutic strategy for increasing the therapeutic effectiveness of anticancer chemotherapies. The general strategy entails expression of an enzyme that catalyzes the breakdown of a prodrug of an anticancer drug thereby localizing the drug in or near the tumor mass and limiting exposure elsewhere. The strategy has been demonstrated using the HSV-TK gene wherein the thymidylate kinase specifically expressed in the transfected cells activates ganciclovir to the monophosphate which is then converted by other kinases to the tumor cell killing triphosphate. A similar strategy uses the bacterial cytosine deaminase gene for conversion of 5-fluorouracil to 5-fluorocytosine. Other genes have been considered including carboxypeptidase G2, nitro reductase, purine nucleoside phosphorylation, etc. In addition, CYP gene transfer has been explored as a way to enhance the chemotherapeutic effect of cyclophosphamide and ifosfamide, two drugs known to be activated by CYPs. For example, human breast cancer cells were sensitized by transfection with the CYP2B1 gene (Chen et al., Cancer Research, 56, 1331 1340 (1996)). The advantage of this strategy relative to the HSV-TK gene strategy is that the product of the CYP catalyzed oxidation readily diffuses outside of the tumor cell and into nearby cells. In contrast to monophosphate products of the HSV-TK strategy, the product can enter cells that are not in cell-cell contact and therefore produce a more widespread tumor killing effect (Chen and Waxman, Cancer Research, 55, 581-589 (1995)).
Compounds of formula 1 can be made more effective by using gene therapy to introduce the gene that encodes the CYP specifically involved in prodrug cleavage. The specific CYP that breaks down the prodrug is readily determined using some or all of the following steps: 1) demonstrate prodrug cleavage using human microsomes; 2) classify the subfamily by comparing activity with microsomes induced with various subfamily specific inducers (e.g. CYP3 enzymes are induced with a variety of steroids, e.g. dexamethasone, antibiotics, e.g. rifampicin, and compounds such as pregnenolone-16alpha carbonitrile, phenytoin, carbamazepine, phenylbutazone, etc.; 3) identify the CYP or CYPs responsible for prodrug activation by using known CYP subfamily specific inhibitors (e.g. troleandomycin, erythromycin, ketoconazole and gestodene) and/or by using neutralizing antibodies; 4) confirm CYP subfamily by demonstrating turnover via the recombinant enzyme.
Genes are introduced to the tumor using a suitable vector (e.g. retroviral vectors, adenoviral vectors) or via direct DNA injection. In theory genes could be introduced into cells which are transplanted into the tumor mass. The compounds of formula 1 are then introduced following significant enhancement of the CYP activity in the tumor.
Potential Non-Cancer Uses
A variety of liver diseases are suitable targets for compounds of formula I. For example, hepatitis is treated with a variety of drugs wherein prodrugs of the type shown in formula 1 will exhibit significant advantages either in regards to efficacy, safety, and/or pharmacokinetics. Other diseases include liver fibrosis wherein disease progression may be slowed or stopped with treatment of proline hydroxylase inhibitors, lysine hydroxylase inhibitors, steroids, non-steroids anti-inflammatory agents, Methotrexate, Ursodeoxycholic acid, and Penicillamine (also for Wilson""s disease). Other possible areas that the prodrugs are useful include the delivery of antimalarial agents, various hepatoprotectants, diagnostic agents etc. Other drugs include azathioprine, chlorambucil.
Preferred Compounds
The compounds of the invention are substituted 6-membered cyclic 1,3-propane diester prodrugs of certain phosph(oramid)ates as represented by Formula I: 
wherein:
V, W, and Wxe2x80x2 are independently selected from the group consisting of xe2x80x94H, alkyl, aralkyl, alicyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, 1-alkenyl, and 1-alkynyl; or
together V and Z are connected via an additional 3-5 atoms to form a cyclic group containing 5-7 atoms, optionally 1 heteroatom, substituted with hydroxy, acyloxy, alkoxycarbonyloxy, or aryloxycarbonyloxy attached to a carbon atom that is three atoms from both Y groups attached to the phosphorus; or
together V and Z are connected via an additional 3-5 atoms to form a cyclic group, optionally containing 1 heteroatom, that is fused to an aryl group at the beta and gamma position to the Y attached to the phosphorus;
together V and W are connected via an additional 3 carbon atoms to form an optionally substituted cyclic group containing 6 carbon atoms and substituted with one substituent selected from the group consisting of hydroxy, acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy, and aryloxycarbonyloxy, attached to one of said carbon atoms that is three atoms from a Y attached to the phosphorus;
together Z and W are connected via an additional 3-5 atoms to form a cyclic group, optionally containing one heteroatom, and V must be aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
together W and Wxe2x80x2 are connected via an additional 2-5 atoms to form a cyclic group, optionally containing 0-2 heteroatoms, and V must be aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
Z is selected from the group consisting of xe2x80x94CHR2OH , xe2x80x94CHR2OC(O)R3, xe2x80x94CHR2OC(S)R3, xe2x80x94CHR2OC(S)OR3, xe2x80x94CHR2OC(O)SR3, xe2x80x94CHR2OCO2R3, xe2x80x94OR2, xe2x80x94SR2, xe2x80x94CHR2N3, xe2x80x94CH2aryl, xe2x80x94CH(aryl)OH, xe2x80x94CH(CHxe2x95x90CR22)OH, xe2x80x94CH(Cxe2x89xa1CR2)OH, xe2x80x94R2, xe2x80x94NR22, xe2x80x94OCOR3, xe2x80x94OCO2R3, xe2x80x94SCOR3, xe2x80x94SCO2R3, xe2x80x94NHCOR2, xe2x80x94NHCO2R3, xe2x80x94CH2NHaryl, xe2x80x94(CH2)pxe2x80x94OR12, and xe2x80x94(CH2)pxe2x80x94SR12;
p is an integer 2 or 3;
with the provisos that:
a) V, Z, W, Wxe2x80x2 are not all xe2x80x94H; and
b) when Z is xe2x80x94R2, then at least one of V, W, and Wxe2x80x2 is not xe2x80x94H, alkyl aralkyl, or alicyclic;
c) when Z is CHR2OH, then M is not xe2x80x94NH(lower alkyl), xe2x80x94N(lower alkyl)2, xe2x80x94NH(lower alkylhalide), xe2x80x94N(lower alkylhalide)2 or xe2x80x94N(lower alkyl)(lower alkylhalide); and
d) when V is aryl or substituted aryl, then M is not xe2x80x94O(D) where D is hydrogen, a metal ion or an ammonium ion;
R2 is selected from the group consisting of R3 and xe2x80x94H;
R3 is selected from the group consisting of alkyl, aryl, alicyclic, and aralkyl;
R6 is selected from the group consisting of xe2x80x94H, lower alkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl, and lower acyl;
R12 is selected from the group consisting of xe2x80x94H, and lower acyl;
each Y is independently selected from the group consisting of xe2x80x94Oxe2x80x94, and xe2x80x94NR6xe2x80x94;
M is selected from the group consisting of drugs MH containing an xe2x80x94OH, xe2x80x94NHR2, or xe2x80x94SH group, and that is attached to the phosphorus in formula I via O, N, or S of said OH, xe2x80x94NHR2, or SH group;
and pharmaceutically acceptable prodrugs and salts thereof.
In general, preferred substituents, V, Z, W, and Wxe2x80x2 of formula I are chosen such that they exhibit one or more of the following properties:
(1) enhance the oxidation reaction since this reaction is likely to be the rate determining step and therefore must compete with drug elimination processes.
(2) enhance stability in aqueous solution and in the presence of other non-P450 enzymes;
(3) enhance cell penetration, e.g. substituents are not charged or of high molecular weight since both properties can limit oral bioavailability as well as cell penetration;
(4) promote the xcex2-elimination reaction following the initial oxidation by producing ring-opened products that have one or more of the following properties:
a) fail to recyclize;
b) undergo limited covalent hydration;
c) promote xcex2-elimination by assisting in the proton abstraction;
d) impede addition reactions that form stable adducts, e.g. thiols to the initial hydroxylated product or nucleophilic addition to the carbonyl generated after ring opening; and
e) limit metabolism of reaction intermediates (e.g. ring-opened ketone);
(5) lead to a non-toxic and non-mutagenic by-product with one or more of the following characteristics. Both properties can be minimized by using substituents that limit Michael additions, e.g.:
a) electron donating Z groups that decrease double bond polarization;
b) W groups that sterically block nucleophilic addition to the xcex2-carbon;
c) Z groups that eliminate the double bond after the elimination reaction either through retautomerization (enol- greater than keto) or hydrolysis (e.g. enamine);
d) V groups that contain groups that add to the xcex1,xcex2-unsaturated ketone to form a ring;
e) Z groups that form a stable ring via Michael addition to double bond; and
f) groups that enhance detoxification of the by-product by one or more of the following characteristics:
(i) confine to liver; and
(ii) make susceptible to detoxification reactions (e.g. ketone reduction); and
(6) capable of generating a pharmacologically active product.
Suitable alkyl groups include groups having from 1 to about 20 carbon atoms. Suitable aryl groups include groups having from 1 to about 20 carbon atoms. Suitable aralkyl groups include groups having from 2 to about 21 carbon atoms. Suitable acyloxy groups include groups having from 1 to about 20 carbon atoms. Suitable alkylene groups include groups having from 1 to about 20 carbon atoms. Suitable alicyclic groups include groups having 3 to about 20 carbon atoms. Suitable heteroaryl groups include groups having from 1 to about 20 carbon atoms and from 1 to 4 heteroatoms, preferably independently selected from nitrogen, oxygen, and sulfur. Suitable heteroalicyclic groups include groups having from 2 to about twenty carbon atoms and from 1 to 5 heteroatoms, preferably independently selected from nitrogen, oxygen, phosphorous, and sulfur.
Preferably, M is attached via an xe2x80x94OH, or xe2x80x94NHR2 group. In one preferred aspect, M is attached to the phosphorus in formula I via an oxygen atom. In another aspect, it is preferred when M is attached via a nitrogen.
In another preferred aspect, MH is useful for the treatment of diseases of the liver or metabolic diseases where the liver is responsible for the overproduction of a biochemical end product. Preferably, such disease of the liver is selected from the group consisting of hepatitis, cancer, fibrosis, malaria, urate production, and chronic cholecystalithiasis. It is more preferred when treating such diseases that MH is an antiviral or anticancer agent.
Preferably, the metabolic disease that MH is useful for hyperlipidemia, diabetes, atherosclerosis, and obesity.
In another aspect, it is preferred when the biochemical end product is selected from the group consisting of glucose, cholesterol, fatty acids, and triglycerides.
In compounds of formula I, preferably both Y groups are xe2x80x94Oxe2x80x94; or one Y is xe2x80x94Oxe2x80x94 and one Y is xe2x80x94NR6xe2x80x94. When only one Y is NR6xe2x80x94, preferably the Y closest to W and Wxe2x80x2 is xe2x80x94Oxe2x80x94. Most preferred are prodrugs where both Y groups are xe2x80x94Oxe2x80x94.
More preferred is when V is selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl.
More preferred V groups of formula VI are aryl, substituted aryl, heteroaryl, and substituted heteoaryl. Preferably Y is xe2x80x94Oxe2x80x94. Preferred compounds of formula VI include those in which Z, W, and Wxe2x80x2 are H, and Z is selected from the group of aryl, substituted aryl, heteroaryl, and substituted heteroaryl. Particularly preferred aryl and substituted aryl groups include phenyl, and phenyl substituted with 1-3 halogens. Especially preferred are phenyl, 3,5-dichlorophenyl, 3-bromo-4-fluorophenyl, 3-chlorophenyl, 2-bromophenyl, and 3-bromophenyl.
It is also especially preferred when V is selected from the group consisting of monocyclic heteroaryl and monocyclic substituted heteroaryl containing at least one nitrogen atom. Most preferred is when such heteroaryl and substituted heteroaryl is 4-pyridyl, and 3-bromopyridyl, respectively.
It is also especially preferred when V is selected from the group consisting of heteroaryl and substituted heteroaryl.
Most preferred is when such heteroaryl is 4-pyridyl.
In another aspect, it is preferred when together V and W are connected via an additional 3 carbon atoms to form an optionally substituted cyclic group containing 6 carbon atoms and monosubstituted with one substituent selected from the group consisting of hydroxy, acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy, and aryloxycarbonyloxy attached to one of said additional carbon atoms that is three atoms from a Y attached to the phosphorus. In such compounds, it is more preferred when together V and W form a cyclic group selected from the group consisting of xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94, CH2CH(OCOR3)xe2x80x94CH2xe2x80x94, and xe2x80x94CH2CH(OCO2)R3)xe2x80x94CH2xe2x80x94.
Another preferred V group is 1-alkene. Oxidation by P450 enzymes is known to occur at benzylic and allylic carbons.
In one aspect, preferred V groups include xe2x80x94H, when Z is xe2x80x94CHR2OH, xe2x80x94CH2OCOR3, or xe2x80x94CH2OCO2R3.
In another aspect, when V is aryl, substituted aryl, heteroaryl, or substituted heteroaryl, preferred Z groups include xe2x80x94OR2, xe2x80x94SR2, xe2x80x94CHR2N3, xe2x80x94R2, NR22, xe2x80x94OCOR3, xe2x80x94OCO2R3, xe2x80x94SCOR3, xe2x80x94SCO2R3, xe2x80x94NHCOR2, xe2x80x94NHCO2R3, xe2x80x94CH2NHaryl, xe2x80x94(CH2)pOR12, and xe2x80x94(CH2)pxe2x80x94SR12. More preferred Z groups include xe2x80x94OR2, xe2x80x94R2, xe2x80x94OCOR3, xe2x80x94OCO2R3, xe2x80x94CH3, xe2x80x94NHCOR2, xe2x80x94NHCO2R3, xe2x80x94(CH2)pxe2x80x94OR12, and xe2x80x94(CH2)pxe2x80x94SR12. Most preferred Z groups include xe2x80x94OR2, xe2x80x94H, xe2x80x94OCOR3, xe2x80x94OCO2R3, and xe2x80x94NHCOR2. In one preferred aspect, Z is not xe2x80x94OR2, xe2x80x94SR2, or NR22.
Preferred W and Wxe2x80x2 groups include H, R3, aryl, substituted aryl, heteroaryl, and substituted aryl. Preferably, W and Wxe2x80x2 are the same group. More preferred is when W and Wxe2x80x2 are H.
In one aspect, prodrugs of formula VI are preferred: 
wherein
V is selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl, 1-alkenyl, and 1-alkynyl. More preferred V groups of formula VI are aryl, substituted aryl, heteroaryl, and substituted heteoaryl. Preferably Y is xe2x80x94Oxe2x80x94. Particularly preferred aryl and substituted aryl groups include phenyl and substituted phenyl. Especially preferred are phenyl, 3,5-dichlorophenyl, 3-bromo-4-fluorophenyl, 3-chlorophenyl, 2-bromophenyl, and 3-bromophenyl. Particularly preferred heteroaryl groups include monocyclic substituted and unsubstituted heteroaryl groups. Especially preferred are phenyl, 3,5-dichlorophenyl, 3-bromo-4-fluorophenyl, 3-chlorophenyl, 2-romophenyl, and 3-bromophenyl, 4-pyridyl, and 3-bromopyridyl. Preferably, Z, W, and Wxe2x80x2 are H. Preferred compounds of formula VI include those in which Z is selected from the group of xe2x80x94OR2, xe2x80x94SR2, xe2x80x94R2, xe2x80x94NR22, xe2x80x94OCOR3, xe2x80x94OCO2R3, xe2x80x94SCOR3, xe2x80x94SCO2R3, xe2x80x94NHCOR2, xe2x80x94NHCO2R3, xe2x80x94(CH2)pxe2x80x94OR12, and xe2x80x94(CH2)pxe2x80x94SR12.
In one aspect, the compounds of formula VI preferably have a group Z which is H, alkyl, alicyclic, hydroxy, alkoxy, OC(O)R3, OC(O)OR3, or NHC(O)R2. Preferred are such groups in which Z decreases the propensity of the by-product, vinylaryl ketone to undergo Michael additions. Preferred Z groups are groups that donate electrons to the vinyl group which is a known strategy for decreasing the propensity of xcex1,xcex2-unsaturated carbonyl compounds to undergo a Michael addition. For example, a methyl group in a similar position on acrylamide results in no mutagenic activity whereas the unsubstituted vinyl analog is highly mutagenic. Other groups could serve a similar function, e.g. Z=OR6, NHAc, etc. Other groups may also prevent the Michael addition especially groups that result in removal of the double bond altogether such as Z=xe2x80x94OH, xe2x80x94OC(O)R3, xe2x80x94OCO2R3, and NH2, which will rapidly undergo retautomerization after the elimination reaction. Certain W and Wxe2x80x2 groups are also advantageous in this role since the group(s) impede the addition reaction to the xcex2-carbon or destabilize the product. Another preferred Z group is one that contains a nucleophilic group capable of adding to the xcex1,xcex2-unsaturated double bond after the elimination reaction ie. (CH2)pSH or (CH2)pOH where p is 2 or 3. Yet another preferred group is a group attached to V which is capable of adding to the xcex1,xcex2-unsaturated double bond after the elimination reaction. V groups are preferred that are substituted aryl and substituted heteroaryl wherein the aryl or heteroaryl group has a suitably positioned nucleophile e.g. 2-OH or SH which can add by an intramolecular reaction to the xcex1,xcex2-unsaturated carbonyl. 
In another aspect, prodrugs of formula VII are preferred: 
wherein
Z is selected from the group consisting of: xe2x80x94CHR2OH, xe2x80x94CHR2OCOR3, xe2x80x94CHR2OC(S)R3, xe2x80x94CHR2OCO2R3, xe2x80x94CHR2OC(O)SR3, and xe2x80x94CHR2OC(S)OR3. Preferably Y is xe2x80x94Oxe2x80x94. More preferred Z groups include xe2x80x94CHR2OH, xe2x80x94CHR2OC(O)R3, and xe2x80x94CHR2OCO2R3. Preferably, when Z isxe2x80x94CHR2OH, and W and Wxe2x80x2 are H, then M does not include within its structure adenine, cytosine, guanine, thymine, uracil, 2,6-diamino purine, hypoxanthine, or a compound of the formula: 
wherein Q is independently H, Cl, NHRQ, NRQ2, NHC(O)RQ, N(C(O)RQ)2, OH or NCHN(RQ)2; and
RQ is C1-C20 alkyl, aryl or aralkyl all optionally substituted with hydroxy or halogen. Also preferred are compounds of formula VII in which when Z, W, and Wxe2x80x2 are H and V is aryl, substituted aryl, heteroaryl or substituted heteroaryl, then M does not include within its structure a group of the following formula: 
xe2x80x83wherein:
RA and RB are independently hydrogen, optionally substituted alkyl having from 1 to about 14 carbons, or optionally substituted cycloalkyl having from 3 to about 10 carbons;
Axe2x80x2 is NH or (CH2)k where k is an integer from 0 to 3; and
Bxe2x80x2 is carbonyl or SO2. Also preferred are compounds of formula VII were V, W, and Wxe2x80x2 are H.
In another aspect, prodrugs of formula VIII are preferred: 
wherein
Zxe2x80x2 is selected from the group consisting of xe2x80x94OH, xe2x80x94OC(O)R3, xe2x80x94OCO2 R3, and xe2x80x94OC(O)SR3;
D4 and D3 are independently selected from the group consisting of xe2x80x94H, alkyl, OR2, xe2x80x94OH, and xe2x80x94OC(O)R3; with the proviso that at least one of D4 and D3 are xe2x80x94H. Preferably Y is xe2x80x94Oxe2x80x94. An especially preferred Z group is OH.
In one preferred embodiment, Wxe2x80x2 and Z are xe2x80x94H, W and V are both the same aryl, substituted aryl, heteroaryl, or substituted heteroaryl, and W and V are cis to each other. In another preferred embodiment, Wxe2x80x2 and Z are xe2x80x94H, W and V are both the same aryl, substituted aryl, heteroaryl, substituted heteroaryl, and W and V are trans to each other. Preferably Y is xe2x80x94O.
In another preferred embodiment, W and Wxe2x80x2 are H, V is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, and Z is selected from the group consisting of xe2x80x94H, OR2, and xe2x80x94NHCOR2. More preferred are such compounds where Z is xe2x80x94H. Preferably, when Z, W, and Wxe2x80x2 are H and V is aryl, substituted aryl, heteroaryl or substituted heteroaryl, then M does not include within its structure a group of the following formula: 
wherein:
RA and RB are independently hydrogen, optionally substituted alkyl having from 1 to about 14 carbons, or optionally substituted cycloalkyl having from 3 to about 10 carbons;
Axe2x80x2 is NH or (CH2)k where k is an integer from 0 to 3; and
Bxe2x80x2 is carbonyl or SO2.
Preferably, such compound have M attached via oxygen. Most preferred are such compounds where oxygen is in a primary hydroxyl or a phenolic hydroxyl group. Also more preferred, are those compounds where V is phenyl or substituted phenyl.
In one embodiment of class (2) prodrugs, preferred are those where the oxygen double bonded to the phosphorus is cis to the H that is geminal to V.
Also more preferred, are those compounds where V is an optionally substituted monocyclic heteroaryl containing at least one nitrogen atom. Preferably such compounds have M attached via oxygen. Most preferred are such compounds where said oxygen is in a primary hydroxyl group or a phenolic hydroxyl. Especially preferred are such compounds where V is 4-pyridyl.
In these compounds it is also preferred when MH is selected from the group consisting of the classes Epipodophyllotoxins, Camptothecins, Anthracyclines, Anthrapyrazoles, Combretastatin Analogs, Enediyine antibiotics, and Taxanes.
Preferred Epipodophyllotoxins include Etoposide, Teniposide, NK-611, GL-331, and azatoxin. More preferred are Etoposide and Teniposide. Preferably, the epipodophyllotoxins are attached to the phosphorus via the phenolic hydroxy group.
Preferred Camptothecins include Camptothecin, Topotecan, Irinotecan (CPT-11), Lurtotecan (GI 147211), 9-aminocamptothecin, GG-211, DX-8951F, SKF 107874, and SKF 108025. Preferably, the Camptothecins are attached to the phosphorus via the C-20 hydroxyl which is tertiary hydroxy group on the lactone ring. More preferred Camptothecins include Camptothecin, Topotecan, Irinotecan, Lurtotecan, and 9-aminocamptothecin. Preferably, these compounds are attached via the C-20 hydroxyl group. For Topotecan, it is also preferable to attach to the phosphorus via the phenolic hydroxyl group. Most preferred Camptothecins include Topotecan and Irinotecan.
Preferred Taxanes include paclitaxel, docetaxel, and FCE-28161. Preferably, the taxanes are attached to the phosphorus via a hydroxy group on the side chain or the secondary hydroxy group on the cyclohexyl ring. More preferred Taxanes include paclitaxel.
Preferred combretastatins include combretastain A-4 and the reported (S,S) dioxolane analog (Bioorg. Med. Chem. Lett. 88: 1997-2000 (1998). Preferably, combretastatins are attached to phosphorus via the phenolic hydroxyl.
Preferred anthrapyrazoles include mitoxantrone, piroxantrone, and Losoxantrone. Preferably, the anthrapyrazoles are attached to phosphorus via a primary alcohol, phenolic hydroxy, or amine depending on the structure. In one aspect, it is preferred to attach the anthrapyrazole via a phenolic hydroxyl. In another aspect it is preferred to attach the anthrapyrazole via a primary alcohol. Especially preferred is mitoxantrone attached to phosphorus via a phenolic hydroxyl.
Preferred Anthracyclines include Doxorubicin, Daunorubicin, Idarubicin, Pirarubicin, and Epirubicin. Preferably, the anthracyclines are attached to the phosphorus via an amine on the sugar moiety. In another preferred aspect, the Anthracyclines are attached to the phosphorus via an alcohol or phenolic hydroxy group. More preferred is attaching via the secondary glycosidic hydroxyl or, when present, a primary alcohol (e.g., Doxorubicin). More preferred Anthracyclines are Doxorubicin, Pirarubicin, Epirubicin, and Idarubicin. Especially preferred Anthracyclines include Pirarubicin and Doxorubicin. In another preferred aspect, the Anthracyclines are attached to the phosphorus via an alcohol or phenolic hydroxy group. More preferred is attaching via the secondary glycosidic hydroxyl or, when present, a primary alcohol (e.g., Doxorubicin).
Preferred Enediyne Antibiotics include neocarzinostatin, calicheamicin, and esperamicin. Preferably, the enediyne antibiotics are attached to the phosphorus via a secondary amine on the sugar moiety. In another aspect, the Enediyne Antibiotics are attached to the phosphorus via a glycosidic hydroxyl. More preferred Enediyne Antibiotics include Neocarzinostatin and Calicheamicin, Dynemicin.
Particularly preferred are such compounds where V is selected from the group consisting of phenyl and 4-pyridyl and MH is selected from the group consisting of etoposide.
Also preferred is when MH is selected from the group consisting of etoposide and doxorubicin.
Preferably, oral bioavailability is at least 5%. More preferably, oral bioavailability is at least 10%.
P450 oxidation can be sensitive to stereochemistry which might either be at phosphorus or at the carbon bearing the aromatic group. The prodrugs of the present invention have two isomeric forms around the phosphorus. Preferred is the stereochemistry that enables both oxidation and the elimination reaction. Preferred is the cis-stereochemistry. In contrast, the reaction is relatively insensitive to the group M since cleavage occurred with a variety of phosphate and phosphoramidates. Accordingly, the group M represents a group that as part of a compound of formula I enables generation of a biologically active compound in vivo by conversion to MH via the corresponding Mxe2x80x94PO32xe2x88x92, Mxe2x80x94P(O)(NHR6)2, or Mxe2x80x94P(O)(Oxe2x88x92)(NHR6). The atom in M attached to phosphorus may be O, S or N. The active drug may be MH or a metabolite of Mxe2x80x94H, but not Mxe2x80x94PO32xe2x88x92 or higher order phosphates, useful for treatment of diseases in which the liver is a target organ, including diabetes, hepatitis, liver cancer, liver fibrosis, malaria and metabolic diseases where the liver is responsible for the overproduction of a biochemical end products such as glucose (diabetes), cholesterol, fatty acids and triglycerides (atherosclerosis). Moreover, Mxe2x80x94H may be useful in treating diseases where the target is outside the liver in tissues or cells that can oxidize the prodrug.
Other preferred M groups include drugs useful in treating diabetes, viral infections, liver fibrosis, parasitic infections, and hyperlipidemia. Such anti-diabetic agents do not include FBPase inhibitors. Generally FBPase inhibitors contain a phosphonate or phosph(oramid)ate group to be active. Specifically, M does not include within its structure optionaly substituted 2-aminocarbonylamino-benzimidazoles, optionally substituted 8-aminocarbonylaminopurines, otionally substituted 2-aminocarbonylamino-indoles, or optionally substituted 2-aminocarbonylamino-azaindole as described in WO 98/39343, WO 98/39344, and WO 98/39342, which are incorporated by reference in their entirety.
The preferred compounds of formula VIII utilize a Zxe2x80x2 group that is capable of undergoing an oxidative reaction that yields an unstable intermediate which via elimination reactions breaks down to the corresponding Mxe2x80x94PO32xe2x88x92, Mxe2x80x94P(O)(NHR6)2, or Mxe2x80x94P(O)(Oxe2x88x92)(NHR6). An especially preferred Zxe2x80x2 group is OH. Groups D4 and D3 are preferably hydrogen, alkyl, xe2x80x94OR2, xe2x80x94OCOR3, but at least one of D4 or D3 must be H.
The following compounds and their analogs can be used in the prodrug methodology of the present invention.
Preferred classes of anticancer drugs include:
Epipodophyllotoxins, camptothecins, endiyne antibiotics, taxanes, coformycins, anthracycline glycosides, mytomycin, combretastatin, anthrapyrazoles, and polyamine biosynthesis inhibitors.
Preferred Epipodophyllotoxins include Etoposide, Teniposide, NK-611, GL-331, and azatoxin. More preferred are Etoposide and Teniposide. Preferably, the epipodophyllotoxins are attached to the phosphorus via the phenolic hydroxy group.
Preferred Camptothecins include Camptothecin, Topotecan, Irinotecan (CPT-11), Lurtotecan (GI 147211), 9-aminocamptothecin, GG-211, DX-8951F, SKF 107874, and SKF 108025. Preferably, the Camptothecins are attached to the phosphorus via the C-20 hydroxyl which is tertiary hydroxy group on the lactone ring. More preferred Camptothecins include Camptothecin, Topotecan, Irinotecan, Lurtotecan, and 9-aminocamptothecin. Preferably, these compounds are attached via the C-20 hydroxyl group. For Topotecan, it is also preferable to attach to the phosphorus via the phenolic hydroxyl group. Most preferred Camptothecins include Topotecan and Irinotecan.
Preferred Taxanes include paclitaxel, docetaxel, and FCE-28161. Preferably, the taxanes are attached to the phosphorus via a hydroxy group on the side chain or the secondary hydroxy group on the cyclohexyl ring. More preferred Taxanes include paclitaxel.
Preferred combretastatins include combretastatin A-4 and the reported (S,S) dioxolane analog (Bioorg. Med. Chem. Lett. 88: 1997-2000 (1998). Preferably, combretastatins are attached to phosphorus via the phenolic hydroxyl.
Preferred anthrapyrazoles include mitoxantrone, piroxantrone, and Losoxantrone. Preferably, the anthrapyrazoles are attached to phosphorus via a primary alcohol, phenolic hydroxy, or amine depending on the structure. In one aspect, it is preferred to attach the anthrapyrazole via a phenolic hydroxyl. In another aspect it is preferred to attach the anthrapyrazole via a primary alcohol. Especially preferred is mitoxantrone attached to phosphorus via a phenolic hydroxyl.
Preferably, the epipodophyllotoxins, camptothecins, combretastatins, anthrapyrazoles, and taxanes are linked to M via an oxygen.
In another aspect, other preferred antineoplastic drugs attached via oxygen include coformycin and deoxycoformycin. Preferably, the coformycins are attached to the phosphorus via the 5xe2x80x2-hydroxy on the sugar moiety.
Preferred Anthracyclines include Doxorubicin, Daunorubicin, Idarubicin, Pirarubicin, and Epirubicin. Preferably, the anthracyclines are attached to the phosphorus via an amine on the sugar moiety. In another preferred aspect, the Anthracyclines are attached to the phosphorus via the secondary glycosidic hydroxyl or when present a primary alcohol (e.g., doxorubicin). More preferred Anthracyclines are Doxorubicin, Pirarubicin, Epirubicin, and Idarubicin. Especially preferred Anthracyclines include Pirarubicin and Doxorubicin. In another preferred aspect, the Anthracyclines are attached to the phosphorus via an alcohol or phenolic hydroxy group. More preferred is attaching via the secondary glycosidic hydroxyl or, when present, a primary alcohol (e.g., Doxorubicin).
In another aspect, Elsamitrocin is preferred. Preferably, elsamitrocin is attached to the phosphorus via the glycosidic amino group or the phenol, more preferably the phenol.
Preferred Enediyne Antibiotics include neocarzinostatin, calicheamicin, and esperamicin. Preferably, the enediyne antibiotics are attached to the phosphorus via a secondary amine on the sugar moiety. In another aspect, the Enediyne Antibiotics are attached to the phosphorus via a glycosidic hydroxyl. More preferred Enediyne Antibiotics include Neocarzinostatin and Calicheamicin.
In another aspect, Mitomycin is also preferred. Preferably, the Mitomycins are attached via the aziridine nitrogen.
Preferred Polyamine Biosynthesis Inhibitors include eflornithine. Preferably, the eflornithine is attached to the phosphorus via a primary amine.
Preferably, the anthracyclines, endiyne antibiotics, mitomycin, and polyamine biosynthesis inhibitors are attached via a nitrogen to the phosphorus.
Where drugs (MH) have more than one hydroxy (phenol, alcohol), thiol, and/or amine group, then in one aspect, it is preferred to attach to the group which is a) important for biological activity; and/or b) important for metabolic destruction of the drugs.
In another aspect, it is preferred to attach the phosphorus to an alcohol or an amine on MH. More preferred is attaching to an alcohol.
Preferred alcohol, amine, and thiol containing compounds encompassed by the invention are compounds with alcohols, amines, or thiols that are readily converted to compounds of formula 1. A preferred process for the conversion of an alcohol, amine or thiol to prodrugs of this invention entails reaction of the alcohol, amine, or thiol on the compound or suitably protected compound with a phosphorylating agent Xxe2x80x94P(O)[xe2x80x94YCH(V)CH(Z)CWWxe2x80x2Yxe2x80x94] wherein X is a suitable leaving group (e.g. halogen, dialkyamine, cyclic secondary amine, and aryloxy). Another preferred process entails reaction of the alcohol, amine, or thiol with a Xxe2x80x94P[xe2x80x94YCH(V)CH(Z)CWWxe2x80x2Yxe2x80x94] wherein X is a suitable leaving group (e.g. halogen, dialkyamine, and cyclic secondary amine) followed by oxidation of the resulting phosph(oramid)ite to the phosph(oramid)ate prodrug using a suitable oxidizing agent (e.g. t-butylhydroperoxide, peroxy acids).
Another process entails conversion of the corresponding Mxe2x80x94PO32xe2x88x92, Mxe2x80x94P(O)(NHR6)2, or MP(O)(NHR6)(Oxe2x88x92) to compounds of formula 1. A preferred process for this conversion entails conversion to Mxe2x80x94P(O)LLxe2x80x2 wherein L and Lxe2x80x2 are independently leaving groups (e.g. halogen, dialkyamine, cyclic secondary amine, and aryloxy). Another process entails initial conversion of the alcohol into a leaving group followed by reaction with HOxe2x80x94P(O)[xe2x80x94YCH(V)CH(Z)CWWxe2x80x2Yxe2x80x94] in the presence of a suitable base. In each case, individual diastereomers are prepared either by isolation from a mixture of diasteromers or by using chiral precursors which are preferably generated using a chiral diol HOCH(V)CH(Z)C(W)(Wxe2x80x2)OH or corresponding chiral amino alcohol.
Most preferred are alcohols, amines, or thiols of the parent drug (MH) selected from the group wherein conversion of the alcohol, amine, or thiol to prodrugs of formula 1 results in substantial loss of biological activity prior to prodrug cleavage. Preferred xe2x80x94OH, amine, and xe2x80x94SH positions on drugs, Mxe2x80x94H, are identified through either knowledge of known structure-activity relationships which indicate the importance of the alcohol, amine, or thiol for biological activity or by studies suitable for characterizing the biological activity of the prodrug without conversion to Mxe2x80x94H. (e.g. cells devoid of P450 activity, Example C).
Most preferred are prodrugs that generate in vivo an unstable intermediate Mxe2x80x94PO32xe2x88x92, MP(O)(NHR6)(Oxe2x88x92), or MP(O)(NHR6)2 intermediate which is dephosphorylated by chemical hydrolysis or preferably by an enzyme (e.g. alkaline phosphatase) and more preferably by a phosphatase or amidase or both in the liver. Prodrugs that are converted to Mxe2x80x94H are readily identified by known methods, including by incubation of the prodrug in the presence of enzymes that catalyze the oxidative cleavage (e.g. cytochrome P450s such as CYP3A4) and that catalyze the dephosphorylation (e.g. phosphatases) either together or as a stepwise process. (Example B) Alternatively, Mxe2x80x94H generation can be monitored by incubating the prodrugs in the presence of hepatocytes (Example A.) Many alcohol-containing drugs are known that as the phosphate convert rapidly to the corresponding alcohol.
Preferred alcohols, amines, or thiols (Mxe2x80x94H) are also alcohols, amines, or thiols wherein selective delivery of the drug to the liver provides a significant therapeutic benefit. The benefit is often observed as improved efficacy since greater liver selectivity can result in higher drug levels in the liver at a given dose or relative to drug levels in plasma. Greater efficacy is possible in cases where maximal efficacy is not achieved due to inadequate peak drug levels or due to inadequate exposure to the drug over a period of time. Inadequate exposure may result from side effects which limit the maximal dose. The benefit may also be exhibited as an improved therapeutic index since in many cases administration of drugs either systemically or orally results in high exposure of the drug to organs throughout the body. This exposure can result in certain toxicities and production of certain pharmacological side effects. For example, oncolytic agents often result in destruction of actively proliferating hematopoietic precursor cells which leads to decreased white blood cells and decreased platelet counts which in turn can result in life-threatening infections and hemorrhage. Other common toxicities include cardiac, pulmonary, reproductive function and nervous system toxicities as well as those observed in the gastrointestinal tract, urinary tract, and at the drug injection sites.
Selective breakdown of the drug by the liver, since the liver is the site which has the highest levels of the P450 isoenzymes that catalyze the oxidative cleavage of the prodrugs of formula 1, is envisioned to result in high liver drug concentrations. In some cases, the drug will remain predominantly in the liver due to high protein binding or due to metabolic processes (e.g. glucoronidation reactions) that convert the drug to metabolites that are retained by the liver. In other cases, the drug will diffuse out of the liver and enter the blood stream and subsequently other tissues. Even for drugs that diffuse out of the liver, however, the drug concentration in the liver will be higher than at extrahepatic sites since the drug is rapidly diluted by body fluids after it leaves the liver (e.g. blood and tissues). The liver drug concentrations should therefore be the highest except for drugs that are actively sequestered by other tissues in the body.
More preferred are compounds useful for treating diseases wherein the liver is the site of the disease (e.g. liver cancer, hepatic viral, bacterial and parasitic infections, liver fibrosis) or is an organ that significantly contributes to the disease (e.g. diabetes, atherosclerosis and obesity). In the latter case, elevated glucose levels in diabetic patients in the postabsorptive state are attributed to the overproduction of glucose by the liver. Elevated lipids and lipid precursors, e.g. cholesterol, fatty acids, triglycerides, and lipid particles are thought to contribute significantly to certain disease conditions such as atherosclerosis and associated heart and other organ diseases as well as obesity. Prodrugs of the invention can be of significant benefit in treating these diseases since the drug levels in the liver are higher than the levels attained after treatment with the drug itself. In other cases, the drug levels in the liver relative to other tissues or blood are higher than after treating with the drug itself. Either profile provides benefit either in the form of enhanced efficacy or improved therapeutic window.
Drugs containing a suitable alcohol include drugs in the epipodophyllotoxin, camptothecin, enediyne, taxane, Elsamitrucin, combretastatin, anthrapyrazole, anthracyclines, and coformycin, classes of anticancer drugs and analogs described in the literature.
Drugs containing a suitable amine include but are not limited to doxorubicin, pirarubicin, mitomycin, methotrexate, enediyne, omithine analogs useful as polyamine biosynthesis inhibitors, and analogs described in the literature.
Drugs containing suitable thiol include but are not limited to penicillamine.
The following prodrugs are preferred compounds of the invention. The compounds are shown without depiction of stereochemistry since the compounds are biologically active as the diastereomeric mixture or as a single stereoisomer. Compounds named in Table 1 are designated by numbers assigned to the variables of formula VIa using the following convention: M1.V.L1.L2 where Y is oxygen and Yxe2x80x2 is oxygen. M1 is a variable that represents compounds of the formula Mxe2x80x94H which have a specific hydroxyl group that is phosphorylated with a group P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) to make compounds of formula V1a. V is an aryl or heteroaryl group that has 2 substituents, L1 and L2, at the designated positions. 
Variable M1 is divided into four groups with the structures assigned to each group listed below:
Variable M1: Group M11:
1) Etoposide where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the 4xe2x80x2 phenolic hydroxyl.
2) Teniposide where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the 4xe2x80x2 phenolic hydroxyl.
3) NK-611 where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the 4xe2x80x2 phenolic hydroxyl.
4) Camptothecin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the C20 hydroxyl.
5) Irinotecan where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the C20 hydroxyl.
6) 9-Aminocamptothecin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the C20 hydroxyl.
7) GG-211 where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the C20 hydroxyl
8) Topotecan where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the C20 hydroxyl
9) Topotecan where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl.
Variable M1: Group M12:
1) Paclitaxel where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the C2xe2x80x2 hydroxyl.
2) Mitoxantrone where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the primary hydroxyl.
3) Combretastatin A-4 where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl
4) Azatoxin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl.
5) Mycophenolic acid where xe2x80x94P(O)Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl.
6) Coformycin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the ribofuranosyl 5xe2x80x2 hydroxyl
7) Mitoxantrone where xe2x80x94P(O)Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl.
8) Paclitaxel where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl in the phenyl propronate prodrugs of BMS180661
9) (S,S) dioxolane derivative of Combretastatin A4 where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl.
Variable M1: Group M13:
1) Doxorubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the glycosidic primary amino group.
2) Daunorubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the glycosidic primary amino group.
3) Idarubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the glycosidic primary amino group.
4) Epirubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the glycosidic primary amino group.
5) Mitomycin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to aziridine amino group.
6) Eflornithine where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the sidechain amino group.
7) 9-Aminocamptothecin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenyl primary amino group.
8) Piroxantrone where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the primary amino group.
9) Calicheamicin theta(I)1 where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the glycosidic primary amino group.
Variable M1: Group M14:
1) Doxorubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the glycosidic hydroxyl group.
2) Pirarubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the glycosidic amino group.
3) Pirarubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the primary C14 hydroxyl group.
4) Pirarubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl furthest from the glycosidic residue.
5) Pirarubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl of N-(4-(hydroxy)phenylacetyl)pirarubicin.
6) Doxorubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the primary hydroxyl group.
7) Doxorubicin where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl of N-(4-(hydroxy)phenylacetyl)pirarubicin
8) Losoxantrone where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl.
9) Irinotecan where xe2x80x94P(O)(Yxe2x80x94CH(V)CH2CH2-Yxe2x80x2) is attached to the phenolic hydroxyl of (4-(hydroxy)phenylacetyl) which is attached to the C20 hydroxyl.
I. VARIABLE V: GROUP V1
1) 2-(L1)-3(L2)phenyl
2) 2-(L1)-4(L2)phenyl
3) 2-(L1)-5(L2)phenyl
4) 2-(L1)-6(L2)phenyl
5) 3-(L1)-4(L2)phenyl
6) 3-(L1)-5(L2)phenyl
7) 3-(L1)-6(L2)phenyl
8) 2-(L1)-6(L2)-4-chlorophenyl
9) 3-(L1)-5(L2) 4-chlorophenyl
II. VARIABLE V: GROUP V2
1) 2-(L1)-3(L2) 4-pyridyl
2) 2-(L1)-5(L2) 4-pyridyl
3) 2-(L1)-6(L2) 4-pyridyl
4) 3-(L1)-5(L2) 4-pyridyl
5) 3-(L1)-6(L2) 4-pyridyl
6) 2-(L1)-4(L2) 3-pyridyl
7) 2-(L1)-5(L2) 3-pyridyl
8) 2-(L1)-6(L2) 3-pyridyl
9) 4-(L1)-5(L2) 3-pyridyl
III. VARIABLE V: GROUP V3
1) 4-(L1)-6(L2) 3-pyridyl
2) 5-(L1)-6(L2) 3-pyridyl
3) 3-(L1)-4(L2) 2-pyridyl
4) 3-(L1)-5(L2) 2-pyridyl
5) 3-(L1)-6(L2) 2-pyridyl
6) 4-(L1)-5(L2) 2-pyridyl
7) 4-(L1)-6(L2) 2-pyridyl
8) 3-(L1)-4(L2)-2-thienyl
9) 2-(L1)-5(L2) 3-furnayl
IV. VARIABLE L1
1) hydrogen
2) chloro
3) bromo
4) fluoro
5) methyl
6) isopropyl
7) methoxy
8) dimethylamino
9) acetoxy
V. VARIABLE L2
1) hydrogen
2) chloro
3) bromo
4) fluoro
5) methyl
6) isopropyl
7) methoxy
8) dimethylamino
9) acetoxy
Preferred compounds are compounds listed in Table 1 using groups M11 and V1. For example, compound 1.3.6.7 represents structure 1 of group M11, i.e. etoposide; structure 3 of group V1, i.e. 2-(L1)-5-(L2)phenyl; structure 6 of variable L1, i.e. isopropyl; and structure 7 of variable L2, i.e. methoxy. The compound 1.3.6.7. therefore is etoposide with the P(O)[Yxe2x80x94CH(V)CH2CH2Yxe2x80x2] attached to the 4xe2x80x2phenolic hydroxyl group being {[1-(2-i-propyl-5-methoxyphenyl)-1,3-propyl]phosphoryl.
Preferred compounds are also compounds listed in Table 1 using groups M11 and V2.
Preferred compounds are also compounds listed in Table 1 using groups M11 and V3.
Preferred compounds are also compounds listed in Table 1 using groups M12 and V1.
Preferred compounds are also compounds listed in Table 1 using groups M12 and V2.
Preferred compounds are also compounds listed in Table 1 using groups M12 and V3.
Preferred compounds are also compounds listed in Table 1 using groups M13 and V1.
Preferred compounds are also compounds listed in Table 1 using groups M13 and V2.
Preferred compounds are also compounds listed in Table 1 using groups M13 and V3.
Preferred compounds are also compounds listed in Table 1 using groups M14 and V1.
Preferred compounds are also compounds listed in Table 1 using groups M14 and V2.
Preferred compounds are also compounds listed in Table 1 using groups M14 and V3.
Preferred compounds are represented by all of the above named compounds with the exception that Y is NH and Yxe2x80x2 is oxygen.
Preferred compounds are represented by all of the above named compounds with the exception that Y is oxygen and Yxe2x80x2 is NH.
Preferred compounds are represented by all of the above named compounds with the exception that Y and Yxe2x80x2 are NH.
Preferred compounds are represented by all of the above named compounds with the exception that Y is NCH3 and Yxe2x80x2 is oxygen.
Preferred compounds are represented by all of the above named compounds with the exception that Y is oxygen and Yxe2x80x2 is NCH3.
Another group of preferred compounds are named in Table 2 and designated by numbers assigned to the variables of formula I using the following convention: M1.Y/Yxe2x80x2.V/Z/W. The compounds are shown without depiction of stereochemistry since the compounds arc biologically active as the diastereomeric mixture or as a single stereoisomer. M1 is a variable that represents compounds of the formula Mxe2x80x94H which have a specific hydroxyl group that is phosphorylated with a group P(O)[Yxe2x80x94CH(V)CH(Z)CH(W)xe2x80x94Yxe2x80x2] to make compounds of formula I. 
The structures for variable M1 are the same as described above.
VI. VARIABLE Y/Yxe2x80x2
1) Y=Yxe2x80x2=oxygen
2) Y=NH; Yxe2x80x2=oxygen
3) Y=oxygen; Yxe2x80x2=NH
4) Y=NH; Yxe2x80x2=NH
5) Y=Nxe2x80x94CH3; Yxe2x80x2=oxygen
6) Y=oxygen; Yxe2x80x2=NCH3
7) Y=Nxe2x80x94CH2CH3; Yxe2x80x2=oxygen
8) Y=N-phenyl; Yxe2x80x2=oxygen
9) Y=Ni-propyl; Yxe2x80x2=oxygen
Variable V/Z/W: Group V/Z/W1
1) V=phenyl; Z=methyl; W=hydrogen
2) V=3,5-dichlorophenyl; Z=methyl; W=hydrogen
3) V=4-pyridyl; Z=methyl; W=hydrogen
4) V=phenyl; Z=methoxy; W=hydrogen
5) V=3,5-dichlorophenyl; Z=methoxy; W=hydrogen
6) V=4-pyridyl; Z=methoxy; W=hydrogen
7) V=phenyl; Z=hydrogen; W=phenyl
8) V=3,5-dichlorophenyl; Z=hydrogen; W=3,5-dichlorophenyl
9) V=4-pyridyl; Z=hydrogen; W=4-pyridyl
Variable V/Z/W: Group V/Z/W2
1) V=phenyl; Z=NHAc; W=hydrogen
2) V=3,5-dichlorophenyl; Z=NHAc; W=hydrogen
3) V=4-pyridyl; Z=NHAc; W=hydrogen
4) V=phenyl; Z=hydrogen; W=methyl
5) V=3,5-dichlorophenyl; Z=hydrogen; W=methyl
6) V=4-pyridyl; Z=hydrogen; W=methyl
7) V=phenyl; Z=hydroxy; W=hydrogen
8) V=3,5-dichlorophenyl; Z=hydroxy; W=hydrogen
9) V=4-pyridyl; Z=hydroxy; W=hydrogen
Variable V/Z/W: Group V/Z/W3
1) V=hydrogen; Z=CH2OH; W=hydrogen
2) V=hydrogen; Z=CH2OC(O)CH3; W=hydrogen
3) V=hydrogen; Z=CH2OC(O)OCH3; W=hydrogen
4) V=methyl; Z=CH2OH; W=hydrogen
5) V=methyl; Z=CH2OC(O)CH3; W=hydrogen
6) V=methyl; Z=CH2OC(O)OCH3; W=hydrogen
7) Z=hydrogen; V and W=xe2x80x94CH2-CH(OH)CH2-
8) Z=hydrogen; V and W=xe2x80x94CH2-CH(OAc)CH2-
9) Z=hydrogen; V and W=xe2x80x94CH2-CH(OCO2CH2CH3)CH2-
Preferred compounds are compounds listed in Table 2 using groups M11 and V/Z/W1. For example, compound 1.1.3 represents structure 1 of group M1, i.e. etoposide; structure 1 of the variable Y/Yxe2x80x2, i.e. both Y and Yxe2x80x2=oxygen; structure 3 of group V/Z/W1, i.e. V=4-pyridyl, Z=methyl and W=hydrogen. The compound 1.1.3. therefore is etoposide with the P(O)(Oxe2x80x94CH(4-pyridyl)CH(CH3)CH2O) attached to the 4xe2x80x2phenolic hydroxyl.
Preferred compounds are also compounds listed in Table 2 using groups M11 and V/Z/W2.
Preferred compounds are also compounds listed in Table 2 using groups M11 and V/Z/W3.
Preferred compounds are also compounds listed in Table 2 using groups M12 and V/Z/W 1.
Preferred compounds are also compounds listed in Table 2 using groups M12 and V/Z/W 2.
Preferred compounds are also compounds listed in Table 2 using groups M12 and V/Z/W 3.
Preferred compounds are also compounds listed in Table 2 using groups M13 and V/Z/W 1.
Preferred compounds are also compounds listed in Table 2 using groups M13 and V/Z/W 2.
Preferred compounds are also compounds listed in Table 2 using groups M13 and V/Z/W 3.
Preferred compounds are also compounds listed in Table 2 using groups M14 and V/Z/W 1.
Preferred compounds are also compounds listed in Table 2 using groups M14 and V/Z/W 2.
Preferred compounds are also compounds listed in Table 2 using groups M14 and V/Z/W 3.
For best mode purposes, it is believed that the best prodrugs are those where V is phenyl substituted with 1-3 halogens or 4-pyridyl, and Z, W and Wxe2x80x2 are H, and both Y groups are oxygen. More preferred are such prodrugs in which V is selected from the group of 3-chlorophenyl, 3-bromophenyl, 2-bromphenyl, phenyl, and 4-pyridyl. When M is PMEA, at this point in time, the best prodrug is where V is 3-chlorophenyl, and Z, W and Wxe2x80x2 are H, and both Y groups are oxygen.
Synthesis of Compounds of Formula I
Synthesis of the compounds encompassed by the present invention includes: I). synthesis of prodrugs; II). and synthesis of substituted-1,3-diols; III.) synthesis of substituted-1,3-amino alcohols and substituted-1,3-diamines.
I) Synthesis of Prodrugs
The following procedures on the preparation of prodrugs illustrate the general procedures used to prepare the prodrugs of the invention which apply to all hydroxy, thiol, and amine-containing drugs. Prodrugs can be introduced at different stages of synthesis of a drug. Most often they are made at a later stage, because of the general sensitivity of these groups to various reaction conditions. Optically pure prodrugs containing a single isomer at phosphorus center can be made either by separation of the diastereomers by a combination of column chromatography and/or crystallization, or by enantioselective synthesis of chiral activated phosph(oramid)ate intermediates. All the procedures described herein, where Y and Yxe2x80x2 are oxygen, are also applicable for the preparation of the prodrugs when Y and/or Yxe2x80x2 are xe2x80x94NR6 by appropriate substitution or protection of nitrogen.
The preparation of prodrugs is further organized into 1) synthesis via activated P(V) intermediates:, 2) synthesis via activated P(III) intermediates, and 3) miscellaneous methods.
I.1 Synthesis via Activated P(V) Intermediate 
I.1.a Synthesis of Activated P(V) Intermediates
In general, synthesis of phosph(oramid)ate esters is achieved by coupling the amine, thiol, or alcohol of MH, with the corresponding activated phosphate precursor for example, Chlorophosphate (Lxe2x80x2=chloro) addition onto hydroxy or amino or thiol-containing drug molecules is one preferred method for preparation of prodrugs. The activated precursor can be prepared by several well known methods. Chlorophosphates useful for synthesis of the prodrugs are prepared from the substituted-1,3-propanediols (Wissner, et al., J. Med Chem., 1992, 35, 1650). Chlorophosphates are made by oxidation of the corresponding chlorophospholanes (Anderson, et al., J. Org. Chem., 1984, 49, 1304) which are obtained by reaction of the substituted diol with phosphorus trichloride. Alternatively, the chlorophosphate agent is made by treating substituted-1,3-diols with phosphorusoxychloride (Patois, et al., J. Chem. Soc. Perkin Trans. I, 1990, 1577). Chlorophosphate species may also be generated in situ from corresponding cyclic phosphites by treatment with carbon tetrachloride in the presence of a base (Silverburg, et al., Tetrahedron lett., 1996, 37, 771), which in turn can be either made from chlorophospholane or phosphoramidite intermediate by treatment with a mild acid. Phosphorofluridate intermediate prepared either from pyrophosphate or phosphoric acid may also act as precursor in preparation of cyclic prodrugs (Watanabe et al., Tetrahedron Lett., 1988, 29, 5763). Similar procedures can be used where Y and/or Yxe2x80x2 are xe2x80x94NR6xe2x80x94.
Phosph(oramid)ates where Lxe2x80x2=NRRxe2x80x2 are also well known intermediates for the synthesis of phosphate esters. Monoalkyl or dialkylphosphoramidate (Watanabe, et al., Chem Pharm Bull., 1990, 38, 562), triazolophosphoramidate (Yamakage, et al., Tetrahedron ,1989, 45, 5459) and pyrrolidinophosphoramidate (Nakayama, et al., J. Am. Chem. Soc., 1990, 112, 6936) are some of the known intermediates used for the preparation of phosphate esters. Another effective phosphorylating procedure is a metal catalyzed addition of the cyclic chlorophosphate adduct with 2-oxazolone. This intermediate attains high selectivity in phosphorylation of primary hydroxy group in presence of secondary hydroxyl group (Nagamatsu, et al., Tetrahedron Lett., 1987, 28, 2375). These agents are obtained by reaction of a chlorophosphate with the amine or alternatively by formation of the corresponding phosphoramidite followed by oxidation. Similar reactions are possible where Y and/or Yxe2x80x2 are xe2x80x94NR6xe2x80x94.
I.1.b. Synthesis of Chiral Activated Phosph(oramid)ate
Phosphorylation of an enantiomerically pure substituted diol with for example, a commercially available phosphorodichloridate Rxe2x80x94OP(O)Cl2, where RO is a leaving group, preferably aryl substituted with electron withdrawing groups, such as a nitro or a chloro, produces two diastereomeric intermediates that can be separated by a combination of column chromatography and/or crystallization. Such a method may also be utilized in preparing chiral chloro phosphonates. Chiral phosphoramidate intermediates can be obtained by utilization of optically pure amine as the chiral auxiliary. This type of intermediate are known to undergo stereospecific substitution (Nakayama, et al. J. Am. Chem. Soc.,1990, 112, 6936). The relative configuration of the phosphorus atom is easily determined by comparison of the 31P NMR spectra. The chemical shift of the equatorial phosphoryloxy moiety (trans-isomer) is always more upfield than the one of the axial isomer (cis-isomer) (Verkade, et al., J. Org. Chem., 1977, 42, 1549). 
Similar methods are possible where Y and/or Yxe2x80x2 are xe2x80x94NR6xe2x80x94.
I.1.c. Synthesis of Prodrugs Using Activated Phosphates
Coupling of activated phosphates with alcohols or amines (MH) is accomplished in the presence of an organic base. For example, Chlorophosphates synthesized as described in the earlier section react with an alcohol in the presence of a base such as pyridines or N-methylimidazole. In some cases phosphorylation is enhanced by in situ generation of iodophosphate from the chlorophosphate (Stomberg, et al., Nucleosides and Nucleotides., 1987, 5: 815). Phosphoroflouridate intermediates have also been used in phosphorylation reactions in the presence of a base such as CsF or n-BuLi to generate cyclic prodrugs (Watanabe et al., Tetrahedron Lett., 1988, 29, 5763). Phosphoramidate intermediates are shown to couple by transition metal catalysis (Nagamatsu, et al., Tetrahedron Lett., 1987, 28, 2375).
Reaction of the optically pure diastereomer of a phosphoramidate intermediate with the hydroxyl of the drug in the presence of an acid produces the optically pure phosphate prodrug by direct SN2(P) reaction (Nakayama, et al. J. Am. Chem. Soc.,1990, 112, 6936). Alternatively, reaction of the optically pure phosphate precursor with a fluoride source, preferably cesium fluoride or tetrabutylammonium fluoride, produces the more reactive phosphorofluoridate which reacts with the hydroxyl of the drug to give the optically pure prodrug by overall retention of configuration at the phosphorus atom (Ogilvie, et al., J. Am. Chem. Soc.,1977, 99, 1277). Chiral phosphonate prodrugs can be synthesized by either resolution of phosphates (Pogatnic, et al., Tetrahedron Lett., 1997, 38, 3495) or by chirality induction (Taapken, et al., Tetrahedron Lett., 1995, 36, 6659; J. Org. Chem., 1998, 63, 8284).
Similar procedures where Y and/or Yxe2x80x2 are xe2x80x94NR6xe2x80x94 are possible.
I.2 Synthesis Via Phosphite Intermediate P(III) 
I.2.a. Synthesis of Activated P(III) Intermediates
Phosphorylation of hydroxy, thiol, and amino groups is achieved using cyclic 1xe2x80x2,3xe2x80x2-propanyl esters of phosphorylating agents where the agent is at the P(III) oxidation state. One preferred phosphorylating agent is a chloro phospholane (Lxe2x80x2=chloro). Cyclic chlorophospholanes are prepared under mild conditions by reaction of phosphorus trichloride with substituted 1,3-diols (Wissner, et al., J. Med. Chem., 1992, 35, 1650). Alternatively phosphoramidites can be used as the phosphorylating agent (Beaucage, et al., Tetrahedron, 1993, 49, 6123). Appropriately substituted phoshoramidites can be prepared by reacting cyclic chlorophospholanes with N,N-dialkylamine (Perich, et al., Aust. J. Chem., 1990, 43, 1623. Perich, et al., Synthesis, 1988, 2, 142) or by reaction of commercially available dialkylaminophosphorodichloridate with substituted propyl-1,3-diols. Similar procedures may be used where Y and/or Yxe2x80x2 are xe2x80x94NR6xe2x80x94.
I.2.b. Synthesis of Chiral Activated P(III) Intermediate
In the cases where unsymmetrical diols are used, the cyclic phosphite is expected to form a mixture of chiral isomers. When an optically active pure diol is used a chromatographically separable mixture of two stable diastereomers with the leaving group (NRRxe2x80x2) axial and equatorial on the phosphorous atom is expected. Pure diasteromers can usually be obtained by chromatographic separation. Chiral induction may also be attained by utilizing chiral amine precursors.
I.2.c. Synthesis of Prodrugs Using Activated Phosphites
Chlorophospholanes are used to phosphorylate alcohols on drug molecules in the presence of an organic base (e.g., triethylamine, pyridine). Alternatively, the phosphite can be obtained by coupling the drug molecule with a phosphoramidate in the presence of a coupling promoter such as tetrazole or benzimidazolium triflate (Hayakawa et al., J. Org. Chem., 1996, 61, 7996). Phosphite diastereomers may be isolated by column chromatography or crystallization (Wang, et al., Tetrahedron Lett, 1997, 38, 3797; Bentridge et al., J. Am. Chem. Soc., 1989, 111, 3981). Since condensation of alcohols with chlorophospholanes or phosphoramidites is an SN2(P) reaction, the product is expected to have an inverted configuration. This allows for the stereoselective synthesis of cyclic phosphites. Stereospecific synthesis of a thermodynamically more stable phosphite is attained by equilibration (e.g., thermal equilibration) reaction, when a mixture of two phosphites are obtained starting from a chiral diol.
The resulting phosphites are subsequently oxidized to the corresponding phosphate prodrugs using an oxidant such as molecular oxygen or t-butylhydroperoxide (Meier et al., Bioorg. Med. Chem. Lett., 1997, 7, 1577). Oxidation of optically pure phosphites is expected to stereoselectively provide optically active prodrugs (Mikolajczyk, et al., J. Org. Chem., 1978, 43, 2132. Cullis, P. M. J. Chem. Soc., Chem Commun., 1984, 1510, Verfurth, et al., Chem. Ber., 1991, 129, 1627). Hence, a combination of thermodynamic equilibration of phosphite and stereoselective oxidation results in chiral prodrugs by starting from chiral diols. Chiral prodrugs may also be obtained starting from chiral drug and a P(V) or P(III) intermediate, when the drug has an imposing topology at the reaction site and has a certain facial selectivity.
I.3. Miscellaneous Methods
Prodrugs are also prepared from the free acid and substituted 1,3-diols by Mitsunobu reactions (Mitsunobu, Synthesis, 1981, 1; Campbell, J. Org. Chem., 1992, 52: 6331), and other acid coupling reagents including, but not limited to, carbodiimides (Alexander, et al., Collect. Czech. Chem. Commun., 1994, 59: 1853; Casara, et al., Bioorg. Med. Chem. Lett., 1992, 2: 145; Ohashi, et al., Tetrahedron Lett., 1988, 29: 1189; Hoffman, M., Synthesis, 1988, 62), and benzotriazolyloxytris-(dimethylamino)phosphonium salts (Campagne, et al., Tetrahedron Lett., 1993, 34: 6743).
Phosphorylation of an alcohol thiol or an amine is also achieved under Mitsunobu reaction conditions using the cyclic 1xe2x80x2,3xe2x80x2-propanyl ester of phosphoric acid in the presence of triphenylphosphine and diethylazodicarboxylate (Kimura et al., Bull. Chem. Soc. Jpn., 1979, 52, 1191). The procedure can be extended to prepare chiral phosphates from enantiomerically pure phosphoric acids.
Prodrugs can be prepared by an alkylation reaction between the phosphonate corresponding tetrabutylammonium salts and substituted-1,3-diiodo propanes made from 1,3-diols (Farquhar, et al., Tetrahedron Lett., 1995 36, 655). Furthermore, phosphate prodrugs can be made by conversion of drug molecule to the dichloridate intermediate with phosphoryl chloride in presence of triethylphosphite and quenching with substituted-1,3-propane diols (Farquhar et al., J. Org. Chem., 1983, 26, 1153).
Phosphorylation can also be achieved by making the mixed anhydride of the cyclic diester of phosphoric acid and a sulfonyl chloride, preferably 8-quinolinesulfonyl chloride, and reacting the hydroxyl of the drug in the presence of a base, preferably methylimidazole (Takaku, et al., J. Org. Chem., 1982, 47, 4937). In addition, starting from a chiral cyclic diester of a phosphoric acid, obtained by chiral resolution (Wynberg, et al., J. Org. Chem., 1985, 50, 4508), one can obtain optically pure phosphates.
Similar procedures may be used where Y and/or Yxe2x80x2 are xe2x80x94NR6xe2x80x94.
II.1) Synthesis of 1,3-Diols
A variety of synthetic methods are known to prepare the following types of 1,3-diols: a) 1-substituted; b) 2-substituted; and c) 1,2- or 1,3-annulated in their racemic or chiral form. Substitution of V, W, Z groups of formula I, can be introduced or modified either during synthesis of diols or after the synthesis of prodrugs.
II.1) 1-Substituted 1,3-Diols
1,3-Dihydroxy compounds can be synthesized by several well known methods in literature. Aryl Grignard additions to 1-hydroxy propan-3-al give 1-aryl-substituted propan-1,3-diols (path a). This method will enable conversion of various substituted aryl halides to 1-arylsubstituted-1,3-propane diols (Coppi, et al., J. Org. Chem., 1988, 53, 911). Aryl halides can also be used to synthesize 1-substituted propanediols by Heck coupling of 1,3-diox-4-ene followed by reduction and hydrolysis (Sakamoto, et al., Tetrahedron Lett., 1992, 33, 6845). Substituted 1,3-diols can be generated enanatioselective reduction of vinyl ketone and hydoboration or by kinetic resolution of allylic alcohol (path b). Variety of aromatic aldehydes can be converted to 1-substituted-1,3-diols by vinyl Grignard addition followed by hydroboration (path b). Substituted aromatic aldehydes are also utilized by lithium-t-butylacetate addition followed by ester reduction (path e) (Turner., J. Org. Chem., 1990, 55 4744). In another method, commercially available cinnamyl alcohols can be converted to epoxy alcohols under catalytic asymmetric epoxidation conditions. These epoxy alcohols are reduced by Red-A1 to result in enantiomerically pure 1,3-diols (path c) (Gao, et al., J. Org. Chem., 1980, 53, 4081). Alternatively, enantiomerically pure 1,3-diols can be obtained by chiral borane reduction of hydroxyethyl aryl ketone derivatives (Ramachandran, et al., Tetrahedron Lett., 1997, 38 761). Pyridyl, quinoline, isoquinoline propan-3-ol derivatives can be oxygenated to 1-substituted-1,3-diol by N-oxide formation followed by rearrangement in acetic anhydride conditions (path d) (Yamamoto, et al., Tetrahedron, 1981, 37, 1871). Aldol condensation is another well described method for synthesis of the 1,3-oxygenated functionality (Mukaiyama, Org. React., 1982, 28, 203). Chiral substituted diols can also be made by enantioselective reduction of carbonyl compounds, by chiral aldol condensation or by enzyme promoted 
kinetic resolution.
II.2) 2-Substituted 1,3-Diols
Various 2-substituted-1,3-diols can be made from commercially available 2-(hydroxymethyl)-1,3-propane diol. Pentaerythritol can be converted to triol via decarboxylation of diacid followed by reduction (path a) (Werle, et al., Liebigs. Ann. Chem., 1986, 944) or diol-monocarboxylic acid derivatives can also be obtained by decarboxylation under known conditions (Iwata, et al., Tetrahedron lett. 1987, 28, 3131). Nitrotriol is also known to give triol by reductive elimination (path b) (Latour, et al., Synthesis, 1987, 8, 742). The triol can be derivatised by mono acetylation or carbonate formation by treatment with alkanoyl chloride, or alkylchloroformate(path d) (Greene and Wuts, Protective groups in organic synthesis, John Wiley, New York, 1990). Aryl substitution can be affected by oxidation to aldehyde and aryl Grignard additions (path c) Aldehydes can also be converted to substituted amines by reductive amination reaction(path e). 
I.3.c) Cyclic-1,3-diols
Compounds of formula 1 where Vxe2x80x94Z or Vxe2x80x94W are fused by four carbons are made from Cyclohexane diol derivatives. Commercially available cis, cis-1,3,5-cyclohexane triol can be used as is or modified as described in case of 2-substituted propan-1,3-diols to give various analogues. These modifications can either be made before or after ester formation. Various 1,3-cyclohexane diols can be made by Diels-Alder methodology using pyrone as diene (Posner, et al., Tetrahedron Lett., 1991, 32, 5295). Cyclohexyl diol derivatives are also made by nitrile oxide-olefin additions (Curran, et al., J. Am. Chem. Soc., 1985, 107, 6023). Alternatively, cyclohexyl precursors are also made from commercially available quinic acid (Rao, et al., Tetrahedron Lett., 1991, 32, 547.)
III. Synthesis of Substituted 1,3-Hydroxyamines and 1,3-Diamines
A large number of synthetic methods are available for the preparation of substituted 1,3-hydroxyamines and 1,3-diamines due to the ubiquitous nature of these functionalities in naturally occurring compounds. Following are some of these methods organized into: 1. synthesis of substituted 1,3-hydroxy amines; 2. synthesis of substituted 1,3-diamines and 3. Synthesis of chiral substituted 1,3-hydroxyamines and 1,3-diamines.
III.1. Synthesis of Substituted 1,3-hydroxy Amines
1,3-Diols described in the earlier section can be converted selectively to either hydroxy amines or to corresponding diamines by converting hydroxy functionality to a leaving group and treating with anhydrous ammonia or required primary or secondary amines (Corey, et al., Tetrahedron Lett., 1989, 30, 5207: Gao, et al., J. Org. Chem., 1988, 53, 4081). A similar transformation may also be achieved directly from alcohols in Mitsunobu type of reaction conditions (Hughes, D. L., Org. React., 1992, 42).
A general synthetic procedure for 3-aryl-3-hydroxy-propan-1-amine type of prodrug moiety involves aldol type condensation of aryl esters with alkyl nitrites followed by reduction of resulting substituted benzoylacetonitrile (Shih et al., Heterocycles, 1986, 24, 1599). The procedure can also be adapted for formation 2-substitutedaminopropanols by using substituted alkylnitrile. In another approach, 3-aryl-3-amino-propan-1-ol type of prodrug groups are synthesized from aryl aldehydes by condensation of malonic acid in presence of ammonium acetate followed by reduction of resulting substituted xcex2-amino acids. Both these methods enable to introduce wide variety of substitution of aryl group (Shih, et al., Heterocycles., 1978, 9, 1277). In an alternate approach, xcex2-substituted organolithium compounds of 1-amino-1-aryl ethyl dianion generated from styrene type of compounds undergo addition with carbonyl compounds to give variety of W, Wxe2x80x2 substitution by variation of the carbonyl compounds (Barluenga, et al., J.Org. Chem., 1979, 44, 4798).
III.2. Synthesis of Substituted 1,3-diamines
Substituted 1,3-diamines are synthesized starting from variety of substrates. Arylglutaronitriles can be transformed to 1-substituted diamines by hydrolysis to amide and Hoffman rearrangement conditions (Bertochio, et al., Bull. Soc. Chim. Fr, 1962, 1809). Whereas, malononitrile substitution will enable variety of Z substitution by electrophile introduction followed by hydride reduction to corresponding diamines. In another approach, cinnamaldehydes react with hydrazines or substituted hydrazines to give corresponding pyrazolines which upon catalytic hydrogenation result in substituted 1,3-diamines (Weinhardt, et al., J. Med. Chem., 1985, 28, 694). High trans-diastereoselectivity of 1,3-substitution is also attainable by aryl Grignard addition on to pyrazolines followed by reduction (Alexakis, et al., J. Org. Chem., 1992, 576, 4563). 1-Aryl-1,3-diaminopropanes are also prepared by diborane reduction of 3-amino-3-arylacrylonitriles which in turn are made from nitrile substituted aromatic compounds (Dornow, et al., Chem Ber., 1949, 82, 254). Reduction of 1,3-diimines obtained from corresponding 1,3-carbonyl compounds are another source of 1,3-diamine prodrug moiety which allows a wide variety of activating groups V and/or Z (Barluenga, et al., J. Org. Chem., 1983, 48, 2255).
III.3. Synthesis of Chiral Substituted 1,3-hydroxyamines and 1,3-diamines
Enantiomerically pure 3-aryl-3-hydroxypropan-1-amines are synthesized by CBS enantioselective catalytic reaction of xcex2-chloropropiophenone followed by displacement of halo group to make secondary or primary amines as required (Corey, et al., Tetrahedron Lett., 1989, 30, 5207). Chiral 3-aryl-3-amino propan-1-ol type of prodrug moiety may be obtained by 1,3-dipolar addition of chirally pure olefin and substituted nitrone of arylaldehyde followed by reduction of resulting isoxazolidine (Koizumi, et al., J. Org. Chem., 1982, 47, 4005). Chiral induction in 1,3-polar additions to form substituted isoxazolidines is also attained by chiral phosphine palladium complexes resulting in enatioselective formation of amino alcohols (Hori, et al., J. Org. Chem., 1999, 64, 5017). Alternatively, optically pure 1-aryl substituted amino alcohols are obtained by selective ring opening of corresponding chiral epoxy alcohols with desired amines (Canas et al., Tetrahedron Lett., 1991, 32, 6931).
Several methods are known for diastereoselective synthesis of 1,3-disubstituted aminoalcohols. For example, treatment of (E)-N-cinnamyltrichloroacetamide with hypochlorus acid results in trans-dihydrooxazine which is readily hydrolysed to erythro-xcex2-chloro-xcex3-hydroxy-xcex3-phenylpropanamine in high diastereoselectivity (Commercon et al., Tetrahedron Lett., 1990, 31, 3871). Diastereoselective formation of 1,3-aminoalcohols is also achieved by reductive amination of optically pure 3-hydroxy ketones (Haddad et al., Tetrahedron Lett., 1997, 38, 5981). In an alternate approach, 3-amninoketones are transformed to 1,3-disubstituted aminoalcohols in high stereoselectivity by a selective hydride reduction (Barluenga et al., J. Org. Chem., 1992, 57, 1219).
All the above mentioned methods may also be applied to prepare corresponding Vxe2x80x94Z or Vxe2x80x94W annulated chiral aminoalcohols. Furthermore, such optically pure amino alcohols are also a source to obtain optically pure diamines by the procedures described earlier in the section.
Formulations
Dose of the prodrugs of the present invention depend on the activity of the parent drug, the disease being treated, the oral bioavailability of the prodrug, and the physical characteristics of the patient.
Compounds of the invention may be administered orally in a total daily dose of about 0.1 mg/kg/dose to about 100 mg/kg/dose, preferably from about 0.3 mg/kg/dose to about 30 mg/kg/dose. The most preferred dose range is from 0.5 to 10 mg/kg (approximately 1 to 20 nmoles/kg/dose). The use of time-release preparations to control the rate of release of the active ingredient may be preferred. The dose may be administered in as many divided doses as is convenient. When other methods are used (e.g. intravenous administration), compounds may be administered to the affected tissue at a rate from 0.3 to 300 nmol/kg/min, preferably from 3 to 100 nmoles/kg/min. Such rates are easily maintained when these compounds are intravenously administered as discussed below.
For the purposes of this invention, the compounds may be administered by a variety of means including orally, parenterally, by inhalation spray, topically, or rectally in formulations containing pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used here includes subcutaneous, intravenous, intramuscular, and intraarterial injections with a variety of infusion techniques. Intraarterial and intravenous injection as used herein includes administration through catheters. Oral administration is generally preferred.
Pharmaceutical compositions containing the active ingredient may be in any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.
Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.
The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain 20 to 2000 xcexcmol (approximately 10 to 1000 mg) of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions. It is preferred that the pharmaceutical composition be prepared which provides easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 0.05 to about 50 xcexcmol (approximately 0.025 to 25 mg) of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.
As noted above, formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary or paste.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach. This is particularly advantageous with the compounds of formula 1 when such compounds are susceptible to acid hydrolysis.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of a drug.
It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs which have previously been administered; and the severity of the particular 
disease undergoing therapy, as is well understood by those skilled in the art.